Detection of nuclease edited sequences in automated modules and instruments

ABSTRACT

The present disclosure provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.62/718,449, filed 14 Aug. 2018; 62/769,805, filed 20 Nov. 2018;62/735,365, filed 24 Sep. 2018; 62/781,112, filed 18 Dec. 2018;62/779,119, filed 13 Dec. 2018; and 62/841,213, filed 30 Apr. 2019. Thisapplication is also related to U.S. application Ser. No. 16/399,988,filed 30 Apr. 2019; and Ser. No. 16/454,865, filed 26 Jun. 2019, all ofwhich are incorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to automated modules and instruments forscreening, identifying, and selecting live cells that havenuclease-directed edits.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowmanipulation of gene sequence, and hence gene function. The nucleasesinclude nucleic acid-guided nucleases, which enable researchers togenerate permanent edits in live cells. Editing efficiencies in cellpopulations can be high; however, in pooled or multiplex formats theretends to be selective enrichment of cells that have not been edited dueto the lack of the double-strand DNA breaks that occur during theediting process. Double-strand DNA breaks dramatically negatively impactcell viability thereby leading to the enhanced survival of uneditedcells and making it difficult to identify edited cells in the backgroundof unedited cells. In addition, cells with edits that confer growthadvantages or disadvantages can lead to skewed representations fordifferent edits in the population.

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved methods for enriching, identifying and selectingcells that have been edited. The present invention satisfies this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides methods, modules and instruments forautomated high-throughput and extremely sensitive enrichment andselection of cells edited by a nucleic acid-guided nuclease. Someembodiments of the methods take advantage of isolation or substantialisolation, e.g., separating individual cells, providing conditions forediting, and growing the individual cells into clonal colonies.Isolation overcomes growth bias from unedited cells, growth effects fromdifferential editing rates, and growth bias resulting from fitnesseffects of different edits. Indeed, it has been determined that removinggrowth rate bias via isolation and growing colonies from the isolatedcells to saturation or terminal colony size improves the observedediting efficiency by up to 4× or more over conventional methods. Otherembodiments of the methods utilize “cherry picking” or direct selectionof edited colonies, determined by the differential growth rate of editedand unedited cell colonies. Cherry picking colonies using the methodsdescribed herein more than doubles the observed editing efficiency asthe result of isolation. Thus, the combination of isolation and cherrypicking improves observed editing efficiency by up to 98% overconventional methods.

The present disclosure provides instruments, modules and methods toenable automated high-throughput and extremely sensitive screening toidentify edited cells in populations of cells that have been subjectedto nucleic acid-guided nuclease editing. The instruments, modules, andmethods take advantage of isolation or substantial isolation, where theterm “isolation” in this context refers to the process of separatingcells and growing them into clonally-isolated formats. The term“substantial isolation” refers to the process of separating cells in apopulation of cells into “groups” of 2 to 100, or 2 to 50, andpreferably 2 to 10 cells. Isolation (or substantial isolation), followedby an initial period of growth (e.g., incubation), editing, and growthnormalization leads to enrichment of edited cells. Further, certain ofthe instruments, modules, and methods described herein facilitate“cherry picking” of edited cell colonies, allowing for direct selectionof edited cells.

Isolation or substantial isolation assists in overcoming the growth biasfrom unedited cells that occurs under competitive growth regimes such asin bulk liquid culture. Indeed, it has been determined that removinggrowth rate bias via isolation or substantial isolation, incubation,editing and normalization improves the observed editing efficiency by upto 4× (from, e.g., 10% to 40% absolute efficiency at population scale)or more over conventional methods, and further that cherry-pickingcolonies using the methods described herein brings the observed editingefficiency up to 8× (from, e.g., 10% to 80% absolute efficiency atpopulation scale) over conventional methods. In someembodiments—particularly in bacteria—the compositions and methods employinducible guide RNA (gRNA) constructs leading to increased observedtransformation efficiency and automation-friendly control over thetiming and duration of the editing process.

One particularly facile module or device for isolation or substantialisolation is a solid wall device where cells are substantially isolated,grown in a clonal (or substantially clonal) format, edited, and eithernormalization or cherry picking is employed. The solid wall devices ormodules and the uses thereof are described in detail herein. Theinstruments, modules and methods in some embodiments allow fornormalization of edited and unedited cell colonies. Normalization refersto growing colonies of cells—whether edited or unedited—to terminalsize; that is, growing the cells until the cells in the colonies entersenescence due to, e.g., nutrient exhaustion or constrained space forfurther growth. Since unedited cells grow more quickly, unedited cellcolonies will reach terminal size (e.g., senescence) before edited cellcolonies; however, the unedited cell colonies eventually “catch up” insize and senescence. Thus, normalization of cell colonies enriches foredited cells as edited cells get “equal billing” with unedited cells.Additionally, certain of the modules as described herein facilitate“cherry picking” of colonies. Cherry picking allows for direct selectionof edited cells by taking advantage of edit-induced growth delay inedited colonies. Cherry picking can be performed by selectingslow-growing cell colonies, or cherry picking can be performed byeliminating faster-growing cell colonies by, e.g., irradiating thefaster-growing cell colonies in the wells in which they are growing.Cherry picking colonies using the instruments, modules, and methodsdescribed herein may more than double the observed editing efficiency asthe result of isolation or substantial isolation alone.

Certain embodiments of the instruments, modules, and methods provide forenriching for edited cells during nucleic acid-guided nuclease editing,where the methods comprise transforming cells with one or more vectorscomprising a promoter driving expression of a nuclease, a promoterdriving transcription of a guide nucleic acid and a donor DNA sequence;diluting the transformed cells to a cell concentration sufficient tosubstantially isolate the transformed cells on a substrate; growing(e.g., incubating) the substantially isolated cells on the substrate;providing conditions for editing; and either 1) growing the cellcolonies to colonies of terminal size (e.g., normalizing the cellcolonies) and harvesting (pooling) the normalized cell colonies; or 2)monitoring the growth of cells colonies on the substrate and selectingslow-growing colonies (or eliminating faster-growing colonies). In someaspects at least the gRNA is optionally under the control of aninducible promoter.

Thus in some embodiments there is provided: a solid wall isolation,induction and normalization (SWIIN) module comprising: a retentatemember comprising: an upper surface and a lower surface and a first andsecond end, an upper portion of a serpentine channel defined by raisedareas on the lower surface of the retentate member, wherein the upperportion of the serpentine channel traverses the lower surface of theretentate member for about 50% to about 90% of the length and width ofthe lower surface of the retentate member; at least one port fluidicallyconnected to the upper portion of the serpentine channel; and areservoir cover at the first end of the retentate member; a permeatemember disposed under the retentate member comprising: an upper surfaceand a lower surface and a first and second end, a lower portion of aserpentine channel defined by raised areas on the upper surface of thepermeate member, wherein the lower portion of the serpentine channeltraverses the upper surface of the permeate member for about 50% toabout 90% of the length and width of the upper surface of the permeatemember, and wherein the lower portion of the serpentine channel isconfigured to mate with the upper portion of the serpentine channel toform a mated serpentine channel; at least one port fluidically connectedto the lower portion of the serpentine channel; and a first and secondreservoir at the first end of the permeate member, wherein the firstreservoir is fluidically connected to the at least one port in theretentate member and the second reservoir is fluidically connected tothe at least one port in the permeate member; a perforated membercomprising at least 25,000 perforations disposed under and adjacent tothe retentate member; a filter disposed under and adjacent to theperforated member and above and adjacent to the permeate member; and agasket disposed on top of the reservoir cover of the retentate member,wherein the gasket comprises a reservoir access aperture and a pneumaticaccess aperture for each reservoir.

In some aspects of this embodiment, the permeate member furthercomprises ultrasonic tabs disposed on the raised areas on the uppersurface of the permeate member and at the first and second end of thepermeate member, the retentate member further comprises recesses for theultrasonic tabs disposed in the raised areas on the lower surface and atthe first and second end of the retentate member, the ultrasonic tabsare configured to mate with the recesses for the ultrasonic tabs, andthe permeate member, retentate member, where the perforated member andthe filter are coupled together by ultrasonic welding. In other aspectsof this embodiment, the permeate member, retentate member, theperforated member and the filter are coupled together by solventbonding.

In some aspects, the first and second reservoirs are each fluidicallycoupled to a reservoir port into which fluids and/or cells flow from thefirst retentate reservoir into the first retentate port and from thefirst permeate reservoir into the first permeate port and into theserpentine channels in the retentate and permeate members.

In some aspects of this embodiment, the SWIIN module further comprises athird and a fourth reservoir, wherein the third reservoir is 1)fluidically coupled to a second port in the retentate member, 2)fluidically coupled to a reservoir access aperture into which fluidsand/or cells flow from outside the SWIIN module into the thirdreservoir, and 3) pneumatically coupled to a pressure source; andwherein the fourth reservoir is 1) fluidically coupled to a second portin the permeate member, 2) fluidically coupled to a reservoir accessaperture into which fluids and/or cells flow from outside the SWIINmodule into the fourth reservoir, and 3) pneumatically coupled to apressure source.

In some aspects of this embodiment, the perforated member comprises atleast 100,000 perforations, or at least 200,000 perforations, or atleast 250,000 perforations, or at least 300,000 perforations, or atleast 350,000 perforations or at least 400,000 perforations, or at least500,000 perforations or more. The volume of the wells formed by theperforations is from 1 nL to 50 nL, or from 2 nL to 40 nL, or from 3 nLto 25 nL, or from 2 nL to 10 nL.

In some aspects, the retentate member is fabricated from polycarbonate,cyclic olefin co-polymer, or poly(methyl methylacrylate). In someaspects of the SWIIN module, a serpentine channel portion of each of theretentate and permeate members is from 75 mm to 350 mm in length, from50 mm to 250 mm in width, and from 2 mm to 15 mm in thickness, and from150 mm to 250 mm in length, from 100 mm to 150 mm in width, and from 4mm to 8 mm in thickness. In some aspects, the volume of the matedserpentine channel is from 4 to 40 mL, or from 6 mL to 30 mL, or from 10mL to 20 mL. In some aspects, the volume of the first and secondreservoir is from 5 to 50 mL, or from 8 to 30 mL, or from 10 to 20 mL.

In some aspects of the SWIIN module, there is a support on each end ofthe permeate member configured to elevate the permeate and retentatemembers above the at least one port in the in retentate member and theat least one port in the permeate member. Certain embodiments of theSWIIN module further comprise imaging means to detect cells growing inthe wells, and in some aspects, the imaging means is a camera with meansto backlight the serpentine channel portion of the SWIIN. In someaspects, the SWIIN module is part of a SWIIN assembly comprising aheated cover, a heater, a fan, and a thermoelectric control device.

In yet another embodiment there is provided a method for performingenrichment of cells edited by a nucleic acid-guided nuclease,comprising: providing transformed cells at a dilution resulting insubstantially cells in an appropriate liquid growth medium comprising0.25%-6% alginate, wherein the cells comprise nucleic acid-guidednuclease editing components where the gRNA optionally is under thecontrol of an inducible promoter; solidifying the alginate-containingmedium with a divalent cation; allowing the isolated cells to grow for 2to 50 doublings to establish cell colonies; optionally inducingtranscription of the gRNA; allowing the cell colonies to grow to becomenormalized; and liquefying the alginate-containing medium with adivalent cation chelating agent. In some aspects, the nucleicacid-guided nuclease editing components are provided to the cells on twoseparate vectors and in some aspects, the nucleic acid-guided nucleaseediting components are provided to the cells on a single vector, and insome aspects, the cells are bacterial cells, yeast cells, or mammaliancells.

In some aspects of this method embodiment, the percentage of alginate inthe growth medium is 1%-4%, and in some aspects, the percentage ofalginate in the growth medium is 2%-3%.

In some aspects, the inducible promoter driving the gRNA is a promoterthat is activated upon an increase in temperature, and in some aspects,the inducible promoter is a pL promoter, the cells are transformed witha coding sequence for the CI857 repressor, and transcription of the oneor more nucleic acid-guided nuclease editing components is induced byraising temperature of the cells to 42° C.

In some aspects, solidifying the alginate-containing medium is performedwith divalent cations except Mg⁺², and in some embodiments, the divalentcation is Ca⁺². In some aspects, the divalent cation chelating agent(e.g., liquefying agent) is citrate, ethylenediaminetetraacetic acid(EDTA), or hexametaphosphate.

Other embodiments provide an automated stand-alone multi-module cellediting instrument comprising: a housing configured to house all or someof the modules; a receptacle configured to receive cells; a receptacleconfigured to receive editing nucleic acids; a growth module for growingcells; a filtration module for concentrating and rendering cellselectrocompetent; a transformation module configured to introduce theediting nucleic acids into the cells; a singulation and editing moduleconfigured to isolate the transformed cells and allow the editingnucleic acids to edit nucleic acids in the cells wherein the singulationand editing module comprises a device for emulsion formation,comprising: a microfluidic device having an emulsion formation unitincluding a sample well configured to receive cells in aqueous medium; acarrier fluid well configured to receive a fluid that is immiscible withthe cells in aqueous medium; a collection substrate to collect aqueousdroplets formed in the immiscible fluid; a sample channel extending fromthe sample well to a channel intersection; a carrier fluid channelextending from the carrier fluid well to the channel intersection; adroplet channel extending from the channel intersection to thecollection substrate; and a pneumatic assembly having a pressure sourceand a pressure sensor, wherein the pneumatic assembly is configured (a)to apply pressure to the emulsion formation unit to drive generation ofdroplets at the channel intersection of the emulsion formation unit andcollect droplets in the collection substrate, (b) to monitor thepressure with the pressure sensor, and (c) to stop application of thepressure to the emulsion formation unit when the pressure sensor detectsa change in pressure indicative of air entering the sample channel fromthe sample well; a processor configured to operate the automatedmulti-module cell editing instrument based on user input and/orselection of a pre-programmed script; and an automated liquid handlingsystem to move liquids from the cell receptacle to the growth module,from the growth module to the filtration module, from the filtrationmodule to the transformation module, from the nucleic acid receptacle tothe transformation module, and from the transformation module to thesingulation and editing module without user intervention.

In some aspects of this embodiment the singulation and editing modulefurther comprises a detection station downstream from the channelintersection and before the collection substrate, in some aspects, thedetection station comprises a camera. In some aspects, the singulationand editing module further comprises a temperature-controlled editingreservoir positioned between the channel intersection and the detectionstation. In some aspects, the detection station detects the opticaldensity of cells in the aqueous droplets, and in some aspects thesingulation and editing module further comprises a droplet sorterpositioned between the detection station and the collection substratewhere the aqueous droplets are sorted into two receptables in thecollection substrate. In other aspects, the collection substratecomprises wells, and is configured to collect one droplet per well. Insome aspects, the collection substrate is temperature-controlled, and insome aspects, the singulation and editing module further comprises adetection station configured to detect droplets in the collectionsubstrate.

Yet other embodiments provide a method for isolating and editing cellsin the automated stand-alone multi-module cell editing instrument havinga microfluidic device, comprising the steps of: providing live cells inthe receptacle configured to receive the live cells; providing editingnucleic acids the receptacle configured to receive editing nucleicacids; growing the live cells in a growth module to a desired opticaldensity to produce grown cells; filtering and rendering electrocompetentthe grown cells to produce filtered cells; transforming the filteredcells in a transformation module configured to introduce the editingnucleic acids into the filtered cells to produce transformed cells;generating droplets in the microfluidic device by providing thetransformed cells in an aqueous medium in the sample well; providing thefluid immiscible with the cells in the aqueous medium in the carrierfluid well; flowing the immiscible fluid from the carrier fluid wellthrough the carrier channel to the channel intersection; flowing thecells in aqueous medium from the sample well through the sample channelto the channel intersection; generating aqueous droplets in theimmiscible fluid; and collecting the aqueous droplets in wells in thecollection substrate; incubating the aqueous droplets in the collectionsubstrate to allow the editing nucleic acids to edit the transformedcells; pooling the aqueous droplets; and using an automated liquidhandling system to 1) transfer the editing nucleic acids from receptacleconfigured to receive nucleic acids to the transformation module, 2)transfer the live cells from the receptacle configured to receive thelive cells to the growth module, 3) transfer the grown cells from thegrowth module to the filtration module; 4) transfer the filtered cellsfrom the filtration module to the transformation module, 5) transfer thetransformed cells to the sample well, and 6) transfer the cells from thecollection substrate to a vessel without user intervention.

An additional embodiment provides a method for isolating and editingcells in the automated stand-alone multi-module cell editing instrumenthaving a microfluidic device, comprising the steps of: providing livecells in the receptacle configured to receive the live cells; providingediting nucleic acids the receptacle configured to receive editingnucleic acids; growing the live cells in a growth module to a desiredoptical density to produce grown cells; filtering and renderingelectrocompetent the grown cells to produce filtered cells; transformingthe filtered cells in a transformation module configured to introducethe editing nucleic acids into the filtered cells to produce transformedcells; generating droplets in the microfluidic device by providing thetransformed cells in an aqueous medium in the sample well; providing thefluid immiscible with the cells in the aqueous medium in the carrierfluid well; flowing the immiscible fluid from the carrier fluid wellthrough the carrier channel to the channel intersection; flowing thecells in aqueous medium from the sample well through the sample channelto the channel intersection; generating aqueous droplets in theimmiscible fluid; and collecting the aqueous droplets one at a time inwells in the collection substrate; incubating the aqueous droplets inthe collection substrate to allow the editing nucleic acids to edit thetransformed cells; monitoring cell growth in the droplets via thedetection station; sorting the aqueous droplets based on rapidity ofcell growth; pooling the aqueous droplets with slow-growing cells in avessel; and using an automated liquid handling system to 1) transfer theediting nucleic acids from receptacle configured to receive nucleicacids to the transformation module, 2) transfer the live cells from thereceptacle configured to receive the live cells to the growth module, 3)transfer the grown cells from the growth module to the filtrationmodule; 4) transfer the filtered cells from the filtration module to thetransformation module, 5) transfer the transformed cells to the samplewell, and 6) transfer the cells from the collection substrate to avessel without user intervention.

In some aspects of these method embodiments, the fluid immiscible withthe cells in the aqueous medium is decane, and in some aspects, thegenerated aqueous droplets comprise cells in a Poisson distribution. Insome aspects, after the pooling step, filtering the edited cells in thefiltration module.

Also provided herein is an automated multi-module cell editinginstrument comprising: an isolation or singulation module, a housingconfigured to house all of some of the modules; a receptacle configuredto receive cells; one or more receptacles configured to receive nucleicacids; a growth module; a transformation module configured to introducethe nucleic acids into the cells; and a processor configured to operatethe automated multi-module cell editing instrument based on user inputand/or selection of a pre-programmed script.

In some aspects of the automated multi-module cell editing instrument,the transformation module comprises a flow-through electroporationdevice; and in some aspects the automated multi-module cell editinginstrument further comprises a cell concentration module. In someaspects the cell concentration module is a tangential flow filtrationmodule. In some aspects a liquid handling system transfers liquidsbetween the modules. And in some aspects, the automated multi-modulecell processing system performs the processes of growing cells,concentrating and rendering the cells electrocompetent, transforming thecells with nucleic acid-guided nuclease editing components, isolatingthe transformed cells, inducing editing in the isolated cells, andgrowing and enriching the cells, all without human intervention.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a substrate; growing the cells and initiating editing; growingthe induced cells into colonies; and selecting cells from thesubstantially isolated colonies from the substrate or pooling cells fromthe substantially isolated colonies from the substrate, wherein thesubstantially isolated colonies are enriched for edited cells. Inoptional aspects of this method, the gRNA is under the control of aninducible promoter and 1) the cells are allowed to grow from 2-200doublings after isolation, and 2) there is an inducing step after thegrowth step and prior to the editing step.

Other embodiments provide a method for enriching edited cells duringnucleic acid-guided nuclease editing comprising: transforming cells withone or more vectors comprising a promoter driving transcription of acoding sequence for a nuclease, a promoter driving transcription of aguide nucleic acid and a DNA donor sequence; diluting the transformedcells to a cell concentration to substantially isolate the transformedcells on a substrate; growing the cells and allowing the cells to edit;growing the cells to form colonies; and selecting small colonies fromthe substantially isolated colonies from the substrate, wherein thesubstantially isolated colonies are enriched for edited cells. Inoptional aspects of this method, the gRNA is under the control of aninducible promoter and 1) the cells are allowed to grow from 2-200doublings after singulation, and 2) there is an inducing step after thegrowth step and prior to the editing step.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a simplified flow chart of exemplary enrichment methods (100a and 100 b) and a selection method (100 c) that may be performed by anautomated module, either as a stand-alone instrument or as part of anautomated multi-module cell processing instrument. FIG. 1B is a plot ofoptical density vs. time showing the growth curves for edited cells(dotted line) and unedited cells (solid line).

FIGS. 2A-2K depict workflows for enriching, and optionally identifyingand selecting edited cells after nucleic acid-guided nuclease genomeediting where the workflows are performed in an automated module, and,optionally, as part of an automated instrument.

FIGS. 3A-3L depict an automated multi-module instrument and componentsthereof with which the enrichment/selection modules may be used.

FIG. 4A is an embodiment of a rotating growth vial that may be used forgrowing cells in an automated multi-module cell processing instrument,as well as for cell isolation in a bulk gel environment. FIG. 4Billustrates a perspective view of one embodiment of a rotating growthdevice in a cell growth module housing. FIG. 4C depicts a cut-away viewof the cell growth module from FIG. 4B. FIG. 4D illustrates the cellgrowth module of FIG. 4B coupled to LED, detector, and temperatureregulating components.

FIGS. 5A-5H depict one embodiment of a SWIIN module. FIG. 5I depicts theembodiment of the SWIIN module in FIGS. 5A-5H further comprising aheater and a heated cover. FIG. 5J is an exemplary pneumaticarchitecture diagram for the SWIIN module described in relation to FIGS.5A-5I, with the status of the components for the various steps listed inTables 1-3.

FIGS. 6A and 6B are simplified flow charts of methods for enriching orselecting edited cells using an automated multi-module cell processinginstrument that includes an isolation/singulation module. FIGS. 6C-6Dare simplified block diagrams of embodiments of an exemplary automatedmulti-module cell processing instrument comprising an isolation andenrichment module. FIG. 6E is a simplified block diagram of anembodiment of an exemplary automated multi-module cell processinginstrument comprising an isolation and selection module.

FIGS. 7A-7C together show a graphic of an experiment performed todemonstrate that normalization is achieved in bulk cell culture. FIG. 7Dis a graphic of a recursive workflow using bulk gel cell culture withcuring. FIG. 7E is a photograph of E. coli cells expressing greenfluorescent protein in 2.0% alginate and medium that has been solidifiedshowing singulated colonies (left) and a photograph of E. coli cellsexpressing green fluorescent protein in 2.0% alginate and medium afterthe medium has been re-liquified. FIG. 7F is a depiction of a bulk cellculture workflow for automation in anisolation/growth/editing/normalization module utilizing a rotatinggrowth vial (such as that shown in FIG. 4A), which in turn may be a partof a multi-module cell editing instrument.

FIGS. 8A, 8B and 8C depict a graph, table, and two graphs, respectively,of the results obtained from editing experiments performed with liquidcell culture employing no isolation or normalization, but employinginducible editing; bulk cell gel culture employing isolation, inducibleediting, and normalization; solid agar plating (SPP) employingisolation, inducible editing, and normalization; solid agar plating(SPP-Cherry) employing isolation, inducible editing, and cherry picking;and solid agar plating (SPP) employing isolation, inducible editing, andnormalization but without cherry picking and simply scraping thecolonies from the plate and re-plating.

FIG. 9A is a photograph of one embodiment of a perforated member to beused in a solid wall device. FIGS. 9B-9D are photographs of E. colicells isolated (via Poisson distribution) and grown into colonies inmicrowells in a solid wall device with a permeable bottom at low,medium, and high magnification, respectively. FIG. 9E shows the resultsof cell colony normalization for E. coli cells under various conditions.FIG. 9F is a photograph of a solid wall device with a permeable bottomon agar, on which yeast cells have been isolated and grown into clonalcolonies. FIG. 9G presents photographs of yeast colony growth at varioustime points.

FIG. 10 is a graph comparing the percentage of editing obtained for astandard plating protocol (SPP), and replicate samples using twodifferent conditions in a solid wall isolation, incubation, andnormalization device (SWIIN): the first with LB+arabinose; and thesecond with SOB followed by SOB+arabinose.

FIGS. 11A-11C are simplified depictions of the status of pressure andvolume for each reservoir in the SWIIN depicted in relation to FIGS.5A-5I commensurate with the pneumatic diagram of FIG. 5T.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentare intended to be applicable to the additional embodiments describedherein except where expressly stated or where the feature or function isincompatible with the additional embodiments. For example, where a givenfeature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., eds., Genome Analysis: A Laboratory Manual Series (Vols.I-IV) (1999); Weiner, Gabriel, Stephens, eds., Genetic Variation: ALaboratory Manual (2007); Dieffenbach, Dveksler, eds., PCR Primer: ALaboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: AMolecular Cloning Manual (2003); Mount, Bioinformatics: Sequence andGenome Analysis (2004); Sambrook and Russell, Condensed Protocols fromMolecular Cloning: A Laboratory Manual (2006); Stryer, Biochemistry (4thEd.) W.H. Freeman, New York N.Y. (1995); Gait, “OligonucleotideSynthesis: A Practical Approach” (1984), IRL Press, London; Nelson andCox, Lehninger, Principles of Biochemistry 3^(rd) Ed., W. H. FreemanPub., New York, N.Y. (2000); Berg et al., Biochemistry, 5^(th) Ed., W.H.Freeman Pub., New York, N.Y. (2002); Doyle & Griffiths, eds., Cell andTissue Culture: Laboratory Procedures in Biotechnology, Doyle &Griffiths, eds., John Wiley & Sons (1998); G. Hadlaczky, ed. MammalianChromosome Engineering—Methods and Protocols, Humana Press (2011); andLanza and Klimanskaya, eds., Essential Stem Cell Methods, Academic Press(2011), all of which are herein incorporated in their entirety byreference for all purposes. CRISPR-specific techniques can be found in,e.g., Appasani and Church, Genome Editing and Engineering From TALENsand CRISPRs to Molecular Surgery (2018); and Lindgren and Charpentier,CRISPR: Methods and Protocols (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”and/or “outer” that may be used herein merely describe points ofreference and do not necessarily limit embodiments of the presentdisclosure to any particular orientation or configuration. Furthermore,terms such as “first,” “second,” “third,” etc., merely identify one of anumber of portions, components, steps, operations, functions, and/orpoints of reference as disclosed herein, and likewise do not necessarilylimit embodiments of the present disclosure to any particularconfiguration or orientation.

Additionally, the terms “approximately,” “proximate,” “minor,” andsimilar terms generally refer to ranges that include the identifiedvalue within a margin of 20%, 10% or preferably 5% in certainembodiments, and any values therebetween.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotides thatare hydrogen bonded to one another with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds. In general, a nucleicacid includes a nucleotide sequence described as having a “percentcomplementarity” or “percent homology” to a specified second nucleotidesequence. For example, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. For example, the donor DNAwill have at least one region of sequence homology (e.g., one homologyarm) to the genomic target locus. In many instances and preferably, thedonor DNA will have two regions of sequence homology (e.g., two homologyarms) to the genomic target locus. Preferably, an “insert” region or“DNA sequence modification” region—the nucleic acid modification thatone desires to be introduced into a genome target locus in a cell—willbe located between two regions of homology. The DNA sequencemodification may change one or more bases of the target genomic DNAsequence at one specific site or multiple specific sites. A change mayinclude changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 or more base pairs of the target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 ormore base pairs of the target sequence. The donor DNA optionally furtherincludes an alteration to the target sequence, e.g., a PAM mutation,that prevents binding of the nuclease at the PAM or spacer in the targetsequence after editing has taken place.

As used herein, “enrichment” refers to enriching for edited cells byisolation or substantial isolation of cells, initial growth of cellsinto cell colonies (e.g., incubation), editing (optionally induced,particularly in bacterial systems), and growing the cell colonies intoterminal-sized colonies (e.g., saturation or normalization of colonygrowth). As used herein, “cherry picking” or “selection of edited cells”refers to the process of using a combination of isolation or substantialisolation, initial growth of cells into colonies (incubation), editing(optionally induced, particularly in bacterial systems), then using cellgrowth—measured by colony size, concentration of metabolites or wasteproducts, or other characteristics that correlate with the rate ofgrowth of the cells—to select for cells that have been edited based onediting-induced growth delay. Selection may entail picking or selectingslow-growing cell colonies; alternatively, selection may entaileliminating (by, e.g., eradicating or removing) the faster-growing cellcolonies.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

As used herein, the terms “isolation” or “isolate” (and “singulation” or“singulate”) mean to separate individual cells so that each cell (andthe colonies formed from each cell) will be separate from other cells;for example, a single cell in a single microwell, or 100 single cellseach in its own microwell. “Isolation” or “isolated cells” result in oneembodiment, from a Poisson distribution in arraying cells. The terms“substantially isolated”, “largely isolated”, and “substantialisolation” (and “substantially singulated”) mean cells are largelyseparated from one another, in small groups or batches. That is, when 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or up to 50—but preferably 10 or lesscells—are delivered to a microwell. “Substantially isolated” or “largelyisolated” result, in one embodiment, from a “substantial Poissondistribution” in arraying cells. With more complex libraries of edits—orwith libraries that may comprise lethal edits or edits withgreatly-varying fitness effects—it is preferred that cells be isolatedvia a Poisson distribution.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible. In the methods describedherein optionally the promoters driving transcription of the gRNAs isinducible.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampilcillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418may be employed. In other embodiments, selectable markers include, butare not limited to human nerve growth factor receptor (detected with aMAb, such as described in U.S. Pat. No. 6,365,373); truncated humangrowth factor receptor (detected with MAb); mutant human dihydrofolatereductase (DHFR; fluorescent MTX substrate available); secreted alkalinephosphatase (SEAP; fluorescent substrate available); human thymidylatesynthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+cells); CD24 cell surfaceantigen in hematopoietic stem cells; rhamnose; human CAD gene to conferresistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drugresistance-1 (MDR-1; P-glycoprotein surface protein selectable byincreased drug resistance or enriched by FACS); human CD25 (IL-2α;detectable by MAb-FITC); Methylguanine-DNA methyltransferase (MGMT;selectable by carmustine); and Cytidine deaminase (CD; selectable byAra-C). “Selective medium” as used herein refers to cell growth mediumto which has been added a chemical compound or biological moiety thatselects for or against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, YACs, BACs, mammalian synthetic chromosomes,and the like. As used herein, the phrase “engine vector” comprises acoding sequence for a nuclease—optionally under the control of aninducible promoter—to be used in the nucleic acid-guided nucleasesystems and methods of the present disclosure. The engine vector mayalso comprise, in a bacterial system, the λ Red recombineering system oran equivalent thereto, as well as a selectable marker. As used hereinthe phrase “editing vector” comprises a donor nucleic acid, including analteration to the target sequence which prevents nuclease binding at aPAM or spacer in the target sequence after editing has taken place, anda coding sequence for a gRNA optionally under the control of aninducible promoter (and preferably under the control of an induciblepromoter in bacterial systems). The editing vector may also comprise aselectable marker and/or a barcode. In some embodiments, the enginevector and editing vector may be combined; that is, the contents of theengine vector may be found on the editing vector.

Editing in Nucleic Acid-Guided Nuclease Genome Systems Generally

The present disclosure provides instruments, modules and methods fornucleic acid-guided nuclease genome editing that provide 1) enhancedobserved editing efficiency of nucleic acid-guided nuclease editingmethods, and 2) improvement in screening for and detecting cells whosegenomes have been properly edited, including high-throughput screeningtechniques. In current protocols employing nuclease systems, bulkculture of cells with constitutively-expressed nuclease componentstypically are used to drive high-efficiency editing. However, pooled ormultiplex formats can lead to selective enrichment of cells that are notedited due to the lack of double-strand DNA breaks that occur duringediting.

Presented herein are methods that take advantage of isolation(separating cells and growing them into clonal colonies) and eithernormalization of cell colonies or cherry picking of slow-growingcolonies. Isolation or substantial isolation, incubation, followed byediting (optionally with transcription of a gRNA under the control of aninducible promoter) and normalization overcomes growth bias fromunedited cells, and substituting cherry picking for normalization allowsfor direct selection of edited cells. The instruments, modules, andmethods may be applied to all cell types including, archaeal,prokaryotic, and eukaryotic (e.g., yeast, fungal, plant and animal)cells.

The instruments, modules, and methods described herein employ editingcassettes comprising a guide RNA (gRNA) sequence covalently linked to adonor DNA sequence where, particularly in bacterial systems, the gRNAoptionally is under the control of an inducible promoter (e.g., theediting cassettes are CREATE cassettes; see U.S. Pat. No. 9,982,278,issued 29 May 2019 and Ser. No. 10/240,167, issued 26 Mar. 2019; Ser.No. 10/266,849, issued 23 Apr. 2019; and U.S. Pub. Ser. No. 15/948,785,filed 9 Apr. 2018; Ser. No. 16/275,439, filed 14 Feb. 2019; and Ser. No.16/275,465, filed 14 Feb. 2019, all of which are incorporated byreference in their entirety). The disclosed methods allow for cells tobe transformed, substantially isolated, grown for several to manydoublings (e.g., incubation), after which editing is allowed. Theisolation process effectively negates the effect of unedited cellstaking over the cell population. The combination of substantiallyisolating cells, then allowing for initial growth followed by optionallyinducing transcription of the gRNA (and optionally the nuclease) andeither normalization of cell colonies or cherry picking cells leads to2-250×, 10-225×, 20-200×, 30-175×, 40-150×, 50-100×, or 10-100× gains inidentifying edited cells over prior art methods and allows forgeneration of arrayed or pooled edited cells comprising cell librarieswith edited genomes. Additionally, the methods may be leveraged tocreate iterative editing systems to generate combinatorial libraries ofcells with two to many edits in each cellular genome. Optionally usinginducible gRNA constructs (and in some embodiments, inducible nucleaseconstructs) provides “pulsed” exposure of the cells to active editingcomponents, which 1) allows for the cells to be arrayed (e.g., largelyisolated) prior to initiation of the editing procedure, 2) decreasesoff-target activity, 3) allows for identification of rare cell edits,and 4) enriches for edited cells or permit high-throughput screeningapplications to identify editing activity using cell growth as a proxyfor editing, by, e.g., measuring optical density, colony size, ormetabolic by-products or other characteristics thereby enriching theedited cell population.

The instruments, compositions and methods described herein improveediting systems in which nucleic acid-guided nucleases (e.g., RNA-guidednucleases) are used to edit specific target regions in an organism'sgenome. A nucleic acid-guided nuclease complexed with an appropriatesynthetic guide nucleic acid in a cell can cut the genome of the cell ata desired location. The guide nucleic acid helps the nucleic acid-guidednuclease recognize and cut the DNA at a specific target sequence. Bymanipulating the nucleotide sequence of the guide nucleic acid, thenucleic acid-guided nuclease may be programmed to target any DNAsequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby. In certain aspects, the nucleic acid-guidednuclease editing system may use two separate guide nucleic acidmolecules that combine to function as a guide nucleic acid, e.g., aCRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In otheraspects, the guide nucleic acid may be a single guide nucleic acid thatincludes both the crRNA and tracrRNA sequences or a single guide nucleicacid that does not require a tracrRNA.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA is encoded bya DNA sequence on a polynucleotide molecule such as a plasmid, linearconstruct, or resides within an editing cassette and isoptionally—particularly in bacterial systems—under the control of aninducible promoter.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence (the portion of theguide nucleic acid that hybridizes with the target sequence) is about ormore than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides inlength. In some embodiments, a guide sequence is less than about 75, 50,45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guidesequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or20 nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be transcribed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript. Alternatively, the guide nucleic acids may be transcribedfrom two separate sequences. The guide nucleic acid can be engineered totarget a desired target DNA sequence by altering the guide sequence sothat the guide sequence is complementary to the target DNA sequence,thereby allowing hybridization between the guide sequence and the targetDNA sequence. In general, to generate an edit in the target DNAsequence, the gRNA/nuclease complex binds to a target sequence asdetermined by the guide RNA, and the nuclease recognizes a protospaceradjacent motif (PAM) sequence adjacent to the target sequence. Thetarget sequence can be any polynucleotide (either DNA or RNA) endogenousor exogenous to a prokaryotic or eukaryotic cell, or in vitro. Forexample, the target sequence can be a polynucleotide residing in thenucleus of a eukaryotic cell. A target sequence can be a sequenceencoding a gene product (e.g., a protein) and/or a non-coding sequence(e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid; that is, the editing cassette may be a CREATEcassette (see, e.g., U.S. Pat. No. 9,982,278, issued 29 May 2019 andSer. No. 10/240,167, issued 26 Mar. 2019; Ser. No. 10/266,849, issued 23Apr. 2019; and U.S. Pub. Ser. No. 15/948,785, filed 9 Apr. 2018; Ser.No. 16/275,439, filed 14 Feb. 2019; and Ser. No. 16/275,465, filed 14Feb. 2019, all of which are incorporated by reference in theirentirety). The guide nucleic acid and the donor nucleic acid may be andtypically are under the control of a single (optionally inducible)promoter. Alternatively, the guide nucleic acid may not be part of theediting cassette and instead may be encoded on the engine or editingvector backbone. For example, a sequence coding for a guide nucleic acidcan be assembled or inserted into a vector backbone first, followed byinsertion of the donor nucleic acid. In other cases, the donor nucleicacid can be inserted or assembled into a vector backbone first, followedby insertion of the sequence coding for the guide nucleic acid. In yetother cases, the sequence encoding the guide nucleic acid and the donornucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments and preferably, the sequence encoding the guidenucleic acid and the sequence encoding the donor nucleic acid are bothincluded in the editing cassette.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve target site recognition fidelity,decrease target site recognition fidelity, and increase the versatilityof a nucleic acid-guided nuclease. In certain embodiments, the genomeediting of a target sequence both introduces a desired DNA change to atarget sequence, e.g., the genomic DNA of a cell, and removes, mutates,or renders inactive a proto-spacer (PAM) region in the target sequence;that is, the donor DNA often includes an alteration to the targetsequence that prevents binding of the nuclease at the PAM in the targetsequence after editing has taken place. Rendering the PAM at the targetsequence inactive precludes additional editing of the cell genome atthat target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired target sequenceedit and an altered PAM can be selected using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe target sequence. Cells that did not undergo the first editing eventwill be cut rendering a double-stranded DNA break, and thus will notcontinue to be viable. The cells containing the desired target sequenceedit and PAM alteration will not be cut, as these edited cells no longercontain the necessary PAM site and will continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because the methods disclosed herein allow foridentification of edited cells in a large background of unedited cells,the methods allow for identification of edited cells where the PAM isless than optimal; that is, the methods for identifying edited cellsherein allow for identification of edited cells even if editingefficiency is very low. Additionally, the present methods expand thescope of target sequences that may be edited since edits are morereadily identified, including cells where the genome edits areassociated with less functional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryoticcells can be yeast, fungi, algae, plant, animal, or human cells.Eukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, mouse, rat,rabbit, dog, or non-human mammal including non-human primate. The choiceof nucleic acid-guided nuclease to be employed depends on many factors,such as what type of edit is to be made in the target sequence andwhether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease may be encoded by a DNAsequence on a vector (e.g., the engine vector) and be under the controlof a constitutive or an inducible promoter. Again, at least one of andpreferably both of the nuclease and guide nucleic acid are under thecontrol of an inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., vector or editing (CREATE) cassette) asthe guide nucleic acid. The donor nucleic acid is designed to serve as atemplate for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 30, 35, 40, 45, 50,75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides or more in length.In certain preferred aspects, the donor nucleic acid can be provided asan oligonucleotide of between 40-300 nucleotides, more preferablybetween 50-250 nucleotides. The donor nucleic acid comprises a regionthat is complementary to a portion of the target sequence (e.g., ahomology arm). When optimally aligned, the donor nucleic acid overlapswith (is complementary to) the target sequence by, e.g., about 10, 15,20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In manyembodiments, the donor nucleic acid comprises two homology arms (regionscomplementary to the target sequence) flanking the mutation ordifference between the donor nucleic acid and the target template. Thedonor nucleic acid comprises at least one mutation or alterationcompared to the target sequence, such as an insertion, deletion,modification, or any combination thereof compared to the targetsequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the donornucleic acid. Moreover, there may be more than one, e.g., two, three,four, or more guide nucleic acid/donor nucleic acid cassettes insertedinto an engine vector, where the guide nucleic acids are under thecontrol of separate, different promoters, separate, like promoters, orwhere all guide nucleic acid/donor nucleic acid pairs are under thecontrol of a single promoter. (See, e.g., U.S. Ser. No. 16/275,465,filed 14 Feb. 2019, drawn to multiple CREATE cassettes.) The promoterdriving transcription of the gRNA and the donor nucleic acid (or drivingmore than one gRNA/donor nucleic acid pair) is optionally an induciblepromoter (and in bacterial systems is preferably an inducible promoter)and the promoter driving transcription of the nuclease is optionally aninducible promoter as well.

Inducible editing is advantageous in that substantially or largelyisolated cells can be grown for several to many cell doublings beforeediting is initiated, which increases the likelihood that cells withedits will survive, as the double-strand cuts caused by active editingare largely toxic to the cells. This toxicity results both in cell deathin the edited colonies, as well as a lag in growth for the edited cellsthat do survive but must repair and recover following editing. However,once the edited cells have a chance to recover, the size of the coloniesof the edited cells will eventually catch up to the size of the coloniesof unedited cells (e.g., the process of “normalization” or growingcolonies to “terminal size”; see, e.g., FIG. 1B described infra).

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence renders the PAM site “immune” to the nucleic acid-guidednuclease and protects the target sequence from further editing insubsequent rounds of editing if the same nuclease is used.

The editing cassette also may comprise a barcode. A barcode is a uniqueDNA sequence that corresponds to the donor DNA sequence such that thebarcode can identify the edit made to the corresponding target sequence.The barcode can comprise greater than four nucleotides. In someembodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid designis associated with a different barcode, or, alternatively, eachdifferent cassette molecule is associate with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

Exemplary Workflows for Editing, Enrichment, and Selection of EditedCells

The methods described herein provide enhanced observed editingefficiency of nucleic acid-guided nuclease editing methods as the resultof a combination of isolation or substantial isolation, initial cellgrowth (incubation), editing, and either normalization of the resultingcell colonies or cherry picking slow-growing cell colonies. Thecombination of the isolation or substantial isolation, initial cellgrowth, editing and normalization processes overcomes the growth bias infavor of unedited cells—and the fitness effects of editing (includingdifferential editing rates)—thus allowing all cells “equal billing” withone another. The combination of isolation or substantial isolation,initial cell growth, editing, and cherry picking allows for directselection of edited colonies of cells. The result of the methodsdescribed herein is that even in nucleic acid-guided nuclease systemswhere editing is not optimal—such as in systems where non-canonical PAMsare targeted—there is an increase in the observed editing efficiency;that is, edited cells can be identified even in a large background ofunedited cells. Observed editing efficiency can be improved up to 80% ormore. Isolating, incubating, editing, and normalization of cell coloniesor cherry picking of cell colonies leads to 2-250×, 10-225×, 25-200×,40-175×, 50-150×, 60-100×, or 5-100× gains in identifying edited cellsover prior art methods and allows for the generation of arrayed orpooled edited cells comprising genome libraries. Additionally, theinstruments, modules and methods may be leveraged to create iterativeediting systems to generate combinatorial libraries, identify rare celledits, and enable high-throughput enrichment applications to identifyediting activity.

FIG. 1A shows simplified flow charts for three exemplary methodsdescribed herein, two for enrichment 100 a and 100 b, and one forselection 100 c. Looking at FIG. 1A, method 100 a begins by transformingcells 110 with the components necessary to perform nucleic acid-guidednuclease editing. For example, the cells may be transformedsimultaneously with separate engine and editing vectors; the cells mayalready be expressing the nuclease (e.g., the cells may have alreadybeen transformed with an engine vector or the coding sequence for thenuclease may be stably integrated into the cellular genome) such thatonly the editing vector needs to be transformed into the cells; or thecells may be transformed with a single vector comprising all componentsrequired to perform nucleic acid-guided nuclease genome editing, whichis advantageous when employing curing and recursive rounds of editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell 110. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of particular interest is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Ser. No. 16/147,120, filed 28 Sep. 2019; Ser. No. 16/147,353, filed 28Sep. 2019; Ser. No. 16/426,310, filed 30 May 2019; and Ser. No.16/147,871, filed 30 Sep. 2018; and U.S. Pat. No. 10,323,258, issued 18Jun. 2019, all of which are incorporated by reference in their entirety.If the screening/selection module is one module in an automatedmulti-module cell editing system, the cells are likely transformed in anautomated cell transformation module.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are isolated (e.g.,singulated) 120; that is, the cells are diluted (if necessary) andplated, arrayed, or otherwise arranged so that cells are sequestered orseparated from one another. Isolation can be performed by, e.g., platingcells at a dilution that separates cells (and the clonal colonies thatgrow) from one another. In some embodiments, isolation itself may act asa partition; in other embodiments, cells are diluted so that they may beflowed into wells where the cells are deposited at an average ofone-half cell per well (that is, using solid walls as a partition); instill other embodiments, the cells may be sequestered or separated fromone another in emulsion droplets (that is, using liquid “walls” as apartition); in yet another exemplary embodiment, the cells may besequestered or separated from one another in a three-dimensional gel(that is, e.g., suspending the cells in liquid, causing the liquid tosolidify into a gel) or by puncture into an agar; and in anotherembodiment the cells may be arrayed on functionalized “islands” on asubstrate (that is, using “virtual wells”, e.g., separated culture areasto culture cells). In addition to selecting a mode for isolated growth,one may select a mode for attaining isolated growth such as random(i.e., Poisson) loading of cells using dilution to assure isolation, orone may use specific cell loading or placement techniques (i.e.,super-Poisson) for loading cells.

Once the cells have been isolated in 100 a, the cells are grown intocolonies of terminal size 130; that is, the colonies arising from theisolated cells are grown into colonies to a point where cell growth haspeaked and is normalized or saturated for both edited and uneditedcells. In the embodiment 100 a shown in FIG. 1A, the editing componentsare under the control of a constitutive promoter; thus, editing beginsimmediately (or almost immediately) upon transformation. However, inother embodiments such as shown in 100 b, at least the guide nucleicacid may be under the control of an inducible promoter, in which caseediting may be induced after, e.g., a number of cell doublings. Colonynormalization may be effected by physical constraint (e.g., well wallsor functionalized islands) or nutrient constraint (e.g., as occurs onsolid agar). At this point, the terminal-size colonies are pooled 140by, e.g., scraping colonies from a solid agar plate, or pooling coloniesin liquid medium from wells. Again, because isolation overcomes growthbias from unedited cells or cells exhibiting fitness effects as theresult of edits made, isolation alone enriches the total population ofcells with cells that have been edited; that is, isolation and,preferably, normalization (e.g., growing colonies to terminal size)allows for high-throughput screening of edited cells.

The method 100 b shown in FIG. 1A is similar to the method 100 a in thatcells of interest are transformed 110 with the components necessary toperform nucleic acid-guided nuclease editing. As described above, thecells may be transformed simultaneously with both the engine and editingvectors, the cells may already be expressing the nuclease (e.g., thecells may have already been transformed with an engine vector or thecoding sequence for the nuclease may be stably integrated into thecellular genome) such that only the editing vector needs to betransformed into the cells, or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing. Further, if theenrichment/selection module is one module in an automated multi-modulecell editing instrument, cell transformation may be performed in anautomated transformation module (e.g., a flow-through electroporationdevice) as described in relation to FIGS. 3C and 3D below.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are isolated 120; thatis, the cells are diluted (if necessary) and plated, arrayed, orotherwise arranged so that cells are sequestered or separated from oneanother. Isolation can be performed by, e.g., plating cells at adilution that separates cells (and the colonies that grow) from oneanother, diluting cells so that they may be flowed into wells where thecells are deposited at an average of one-half cell per well, or thecells may be sequestered or separated from one another in emulsiondroplets, or on functionalized islands. Further, depending on the deviceor method used to isolated cells, the cells can be loaded according to aPoisson or super-Poisson distribution.

Once the cells have been isolated 120, the cells are allowed to grow to,e.g., between 2 and 50, or between 5 and 40, or between 10 and 30doublings, establishing clonal colonies 150. After colonies are grown,editing is induced 160 by, e.g., activating one or more induciblepromoters that control transcription of one or more of the componentsneeded for nucleic acid-guided nuclease editing, such as transcriptionof the gRNA or nuclease. Once editing is induced 160, many of the editedcells in the clonal colonies die due to the double-strand DNA breaksthat occur during the editing process and are not repaired; however, ina percentage of edited cells, the genome is edited and the double strandbreak is properly repaired. These edited cells then start growing andre-establish colonies and the colonies are allowed to grow to terminalsize 170. Once the colonies have reached terminal size, the colonies arepooled 140. Thus, methods 100 a and 100 b both allow for enrichment ofedited cells. Method 100 c is drawn to selection of edited cells bycherry picking edited colonies.

The method 100 c shown in FIG. 1A is similar to the methods 100 a and100 b in that cells of interest are transformed 110 with the componentsnecessary to perform nucleic acid-guided nuclease editing. As describedabove, the cells may be transformed simultaneously with both the engineand editing vectors, the cells may already be expressing the nuclease(e.g., the cells may have already been transformed with an engine vectoror the coding sequence for the nuclease may be stably integrated intothe cellular genome) such that only the editing vector needs to betransformed into the cells, or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing. Further, if theenrichment/selection module is one module in an automated multi-modulecell editing instrument, cell transformation may be performed in anautomated transformation module as described in relation to FIGS. 3C and3D below.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are isolated 120; thatis, the cells are diluted (if necessary) and plated, arrayed, orotherwise arranged so that cells are sequestered or separated from oneanother. Isolation can be performed by, e.g., plating cells at adilution that separates cells (and the colonies that grow) from oneanother, diluting cells so that they may be flowed into wells where thecells are deposited at an average of one-half cell per well, or thecells may be sequestered or separated from one another in emulsiondroplets, or on functionalized islands. Further, depending on the deviceor method used to isolate cells, the cells can be loaded according to aPoisson or super-Poisson distribution.

Once the cells have been isolated 120, the cells are allowed to grow to,e.g., between 3 and 200, or between 5 and 150, or between 10 and 100doublings, establishing clonal colonies 150. Again, after colonies aregrown, editing is induced 160 by, e.g., activating one or more induciblepromoters that control transcription of one or more of the componentsneeded for nucleic acid-guided nuclease editing, such as transcriptionof the gRNA or nuclease. Once editing is induced 160, many of the editedcells in the clonal colonies die due to the double strand DNA breaksthat occur during the editing process that are not repaired; however, ina percentage of edited cells the genome is edited and the double strandbreak is properly repaired. These cells in which the break is properlyrepaired then start growing and re-establish colonies and the coloniesare allowed to grow until colonies form. In method 100 c, the growth ofthe cell colonies is monitored via, e.g., size, OD, the concentration ofcell metabolites, etc. to identify colonies of cells where growth lagsbehind other, more rapidly-growing colonies. Once small colonies havebeen identified, the small colonies optionally can be selected or cherrypicked 180 to directly select for edited cells. In one alternative tothis general method 100 c, an inducible promoter is not used and editingis not induced; instead, editing essentially begins immediately upontransformation. Still, cells are allowed to establish colonies, and theslow-growing colonies can be cherry picked.

Another alternative exemplary workflow, cells are transformed first withthe editing vector, then isolated by, e.g., growing colonies and pickingor by going directly into, e.g., microtiter plates. Once isolated, thecells are allowed to build cell mass to survive cutting. At thisjuncture, the engine vector (expressing the nuclease) is transformedinto the cells and the cells are allowed to grow as described above andpooled, or, alternatively, cherry picked. Because isolation eliminatesthe bias of non-editing cells and fitness effects from editing as wellas effects from differential editing rates, isolation alone enriches forediting cells such that all isolated colonies—not only the slow-growingcolonies—may be pooled into an “enriched” edited pool of cells.

FIG. 1B is a plot of optical density versus time showing the growthcurves for edited cells (dotted line) and unedited cells (solid line).Note that there is a growth lag for edited cells; however, eventuallythe growth of the edited cells catches up with the growth of theunedited cells. Herein, this phenomenon is referred to as growth of cellcolonies to “terminal size”, “saturation”, or “normalization.”

Exemplary Workflows for Screening and Selection

The methods described herein provide enhanced observed editingefficiency of nucleic acid-guided nuclease editing methods, andimprovement in enrichment (including high-throughput enrichment) andselection for cells whose genomes have been properly edited. Theexemplary workflows described herein employ the concept of isolation.Isolation overcomes the growth bias in favor of unedited cells—and thefitness effects of editing (including differential editing rates)—thusallowing all cells “equal billing” with one another. Further, selection(e.g., cherry picking) may be performed by taking advantage of thegrowth lag of colonies of edited cells in comparison to colonies ofnon-edited cells. The result of the methods is that even in nucleicacid-guided nuclease systems where editing is not optimal (such as insystems where non-canonical PAMs are targeted), there is an increase inthe observed editing efficiency; that is, edited cells can be identifiedeven in a large background of unedited cells. Observed editingefficiency can be improved up to 98%.

FIGS. 2A-2K depict improved workflows for enrichment and, in someembodiments, identifying and selecting edited cells after nucleicacid-guided nuclease genome editing. FIG. 2A depicts a protocol forhigh-throughput selection using colony morphology to identify editedcells. In edited cells, cell viability is compromised in the periodafter editing begins. The workflow depicted in FIG. 2A takes advantageof the growth lag in colonies of edited cells to identify edited cells.In some embodiments, the colony size of the edited cells is 20% smallerthan colonies of non-edited cells once the colonies of edited cellsbegin to appear to the naked eye. In some aspects, the colony size ofthe edited cells is 30%, 40%, 50%, 60%, 70%, 80% or 90% smaller than thecolonies of non-edited cells once the colonies of edited cells begin toappear to the naked eye. In many embodiments, the colony size of theedited cells is 30-80% smaller than colonies of non-edited cells, and insome embodiments, the colony size of the edited cells is 40-70% smallerthan colonies of non-edited cells once the colonies of edited cellsbegin to appear to the naked eye.

In FIG. 2A, a library or collection of editing vectors 202 is introduced203 (e.g., electroporated) into cultured cells 204 that comprise acoding sequence for a nuclease under the control of a constitutive orinducible promoter, 1) contained on an “engine plasmid” (most oftenalong with a selectable marker) that has already been transformed intothe cells; 2) integrated into the genome of the cells being transformed;or 3) contained or located on the editing vector 202 (e.g., a singlevector system). The “introduction” of nucleic acids into the cells(e.g., transformation or transfection) may be accomplished manuallybefore transferring the cells to an enrichment and selection module, or“introducing” the nucleic acids into the cells may take place in anautomated transformation module as described below in relation to FIGS.3A-3D. The editing vectors 202 comprise a donor nucleic acid editingsequence, a PAM-altering sequence (most often a sequence that disablesthe PAM or spacer at the target site in the genome), a coding sequencefor a gRNA, and a selectable marker. In some embodiments, the gRNA,donor nucleic acid, and optional PAM-altering sequence are all containedon an editing cassette, and are all under the control of (e.g., areoperably linked to) a single promoter; preferably, the single promoteris an inducible promoter.

At step 205, the transformed cells are diluted and plated (e.g.,isolated) onto selective medium 206 (in this case nutrient agar) thatselects for both the engine and editing vectors if two plasmids are used(e.g., medium containing chloramphenicol (engine) and carbenicillin(editing)), or a selective medium 206 for the single combinedengine/editing plasmid if a single plasmid system is used. Once plated,the cells are grown 207 at 30° C. for approximately 2-12 hours so thatthe cells grow to form colonies on plate 208. Once colonies appear,there are large 214 and small 212 colonies. The large colonies 214likely represent cells that have not been edited due to, e.g., aninactive gRNA or nuclease. The colonies with small size 212 are likelycolonies of cells that have been edited as the double-strand cuts causedby active editing without repair are largely toxic to the cells. Thistoxicity results both in cell death in the edited colonies, as well as alag in growth for the edited cells that do survive but must repair andrecover following editing. Here, the small colonies (edited cells) arecherry picked 211 and are arrayed on a 96-well plate 218. Cells in the96-well plate 218 can be cultured, and aliquots from this 96-well plate218 can be sequenced and edited colonies identified. This 96-well platemay be kept as a “cell hotel” or cell repository, and once cells thathave been properly edited are identified by, e.g., sequenceverification, one can retrieve the cells with the desired edit from“cell hotel” plate 218.

Alternatively, small colonies 212 may be picked and pooled 209 foradditional rounds of editing (without sequence verification), as thepopulation of cells that goes through the next round of editing hasenriched and selected for edited cells from the first round of editingby virtue of isolation and cherry picking. The method depicted in FIG.2A employs isolation and cherry picking and thus allows for ahigh-throughput method to identify cells that have a high likelihood tobe edited. Screening out a large proportion of the cells withnon-functional gRNAs or nucleases allows for identification of editedcells more readily. It has been determined that removing growth ratebias via isolation (screening) and growing colonies to terminal sizeimproves the observed editing efficiency by up to 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×, O1× or more over conventional methods where isolation is notemployed, and further that cherry picking (e.g., selection) coloniesusing the methods described herein increases by 1.5×, 1.75×, 2.0×, or2.5× or more the observed editing efficiency due to isolation only. Thecombination of isolation and cherry-picking results in an increase of upto 98% in observed editing efficiency. Example 1 below providesmaterials and methods for this embodiment.

An additional feature of the method depicted in FIG. 2A is that in someembodiments at least the gRNA is under the control of an induciblepromoter, particularly in bacterial systems and in other cells thatdivide rapidly. In this instance, isolation is carried out in the samemanner (e.g., here, plating 205); however, instead of growing the cellsfor 12 hours with continuous editing, the cells are allowed to doubleapproximately to between 2 and 200, or between 5 and 150, or between 10and 100 times to form clonal colonies, then editing is induced by, e.g.,temperature or inducer chemicals (e.g., sugars, antibiotics). Thismethod is discussed in relation to FIG. 1 at method 100 c. Afterinduction of editing, the cells are allowed to grow to continue toestablish colonies. Again, once colonies appear, there are large 214 andsmall 212 colonies. The large colonies 214 represent cells that have notbeen edited due to, e.g., an inactive gRNA or nuclease (e.g.,“escapees”). The colonies with small size 212 are likely colonies ofcells that have been edited as the double-strand cuts caused by activeediting that are not repaired are largely toxic to the cells. Thistoxicity results both in cell death for many of the cells in the editedcolonies, as well as a possible lag in growth for the edited cells thatdo survive but must repair and recover following editing. The smallcolonies (edited cells) may be cherry picked 211 and are arrayed on a96-well plate 218.

The modules implementing the workflow described in FIG. 2A—as well asthe workflows in FIGS. 2B-2I—may employ “off the shelf” liquid handlinginstrumentation such as that sold by Opentrons (OT-2™ system, Brooklyn,N.Y.); ThermoFisher Scientific (Versette™ system, Carlsbad, Calif.);Labcyte (Access™ system, San Jose, Calif.); Perkin Elmer (Janus™ system,San Jose, Calif.); Agilent Inc. (Bravo™ system, Santa Clara, Calif.);BioTek Inc. (Winoosky, Vt.); Hudson Inc. (Solo™ system, Springfield,N.J.); Andrew Alliance (Andrew™ system, Waltham, Mass.); and HamiltonRobotics (suite of tools, Reno, Nev.). Further, in workflow embodimentssuch as those depicted in FIGS. 2A and 2B, automated colony pickers maybe employed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK). Further, colony growth onplates (such as shown in FIGS. 2A and 2B) can be monitored by automateddevices such as those sold by JoVE (ScanLag™ system, Cambridge, Mass.)(also see Levin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cellgrowth for, e.g., mammalian cells may be monitored by, e.g., the growthmonitor sold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLosOne, 11(2):e0148469 (2016)).

While the method for selecting for edited cells using cell growth as aproxy for editing has been described herein in the context of measuringcolony size of cell colonies on a agar plate (such as in FIGS. 2A and2B), the optical density (OD) of growing cell colonies, such as in amicrotiter plate or other substrate with wells or functionalizedregions, a series of tubes, or in droplets may be measured instead (asdescribed in relation to FIGS. 2C and 2G). Additionally, other cellgrowth parameters may be measured in addition to or instead of cellcolony size or OD, particularly if the growth parameters allow a simplereadout by colorimetric (or other optically-detectable) methods. Forexample, spectroscopy using visible, UV, or near infrared (NIR) lightallows monitoring the concentration of nutrients and/or wastes in thecell culture. Moreover, spectroscopic measurements may be used toquantify multiple chemical species simultaneously.

For example, cell proliferation assays can monitor various parameters ofcell growth and functioning. One common exemplary method for assessingcell proliferation is a colorimetric assay based on DNA content incells. New DNA synthesis provides a precise marker that may bemultiplexed with other cellular markers like mitochondrial function orcell morphology. Other colorimetric assays include the BrdU, EdU, MTT,XTT, WST-1, Ki67, CFSE, Live/Dead, Trypan Blue, or β-gal assays. BrdUincorporates into newly-synthesized DNA and is detected using anti-BrdUantibody. EdU is similar to BrdU but EdU employs detection withoutantibodies. MTT, a yellow tetrazole, is reduced to purple formazan inliving cells. The XTT assay is based on the premise that activelyrespiring cells convert XTT to a water-soluble, orange-colored formazan.In the WST-1 assay, WST-1 is cleaved to a soluble formazan by a complexcellular mechanism that occurs at the cell surface. For the Ki67 assay,antibodies to Ki67 nuclear protein can be used to measure cellularproliferation. In yet another colorimetric assay, CFSE, anon-fluorescent cell permeable dye, is cleaved by intracellularesterases resulting in green fluorescence. The live/dead cell assayemploys simultaneous fluorescence staining of viable and dead cellsusing calcein-AM and propidium iodide, which stain viable and deadcells, respectively. In the trypan blue assay, dye exclusion is based onthe concept that viable cells do not take up impermeable dyes but deadcells are permeable. In the β-gal assay, beta-galactosidase enzymeactivity is detectable in senescent cells that do not proliferate.

Alternatively, hyperspectral imaging, such as in the near-infraredrange, may be employed (see Feng, et al., Scientific Reports, 7:15934(2017)), or coherent anti-stokes scattering hyperspectral imaging may beemployed (see, e.g., Masia, et al., Anal. Chem., 90:3775-85 (2018)).Near-infrared imaging also may be applied to nonsymmetric chemicalspecies. Conversely, symmetric chemical species can be readilyquantified using Raman spectroscopy. Many critical metabolites, such asglucose, glutamine, ammonia, and lactate have distinct spectral featuresin the IR, such that they may be easily quantified. The amount andfrequencies of light absorbed by the sample can be correlated to thetype and concentration of chemical species present in the sample. Eachof these measurement types provides specific advantages. FT-NIR providesthe greatest light penetration depth and so can be used for thickersamples so that they provide a higher degree of light scattering.FT-mid-IR (MIR) provides information that is more easily discernible asbeing specific for certain analytes as these wavelengths are closer tothe fundamental IR absorptions. FT-Raman is advantageous when theinterference due to water is to be minimized. Other spectral propertiescan be measured via, e.g., dielectric impedence spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH,and/or conductivity may be used to assess the rate of cell growth.

FIG. 2B depicts additional detail of the exemplary embodiment shown inFIG. 2A. FIG. 2B shows high-throughput selection 220 using colonymorphology to identify and cherry pick edited cells. As described above,in edited cells cell viability is compromised in the period afterediting is induced. The present method takes advantage of cell isolationand the growth lag in colonies of edited cells to identify edited cells.In FIG. 2B, transformed cells are diluted and plated on mediumcontaining arabinose 208 and grown for a period of time at, e.g., 30° C.Thus, the workflow shown in FIG. 2B, like the workflow shown in FIG. 2A,employs dilution of cells to achieve isolation, as opposed to wells,droplets, or functionalized islands. After enough time for colonies tobegin to form (e.g., approximately between 2 and 50, or between 5 and40, or between 10 and 30 cell doublings), editing is induced by, e.g.,activating an inducible promoter. In this model system, a library ofediting oligos each configured to inactivate galK is exemplified.Successful editing of cells with the galK editing oligos results inwhite (open circles)—as opposed to red (filled circles)—colonies whenplated on MacConkey agar. Colonies are allowed to grow and both small212 and large 214 colonies result.

Colonies from plate 208 are picked 221 and arrayed on a second plate 222containing McConkey agar, e.g., a medium to select for successfulediting of galK. Note that picking small colonies 212 from the firstplate results primarily in edited cells 226 (white colonies) and—at amuch lower frequency—some cells in which the gRNA or nuclease isinactive 224 (red colonies, shown here as filled-in circles).Confirmation of colonies in which the gRNA is inactive is shown bypicking 225 large colonies 214 from the first plate and plating them onthe second plate 222 where these cells result in red colonies 224 whengrown on MacConkey agar supplemented with galactose as the sole carbonsource. Thus, using small and large colony morphology as a proxy foredited and non-edited cells, respectively, provides a high throughputand facile screening method for edited cells. The methods depicted inFIGS. 2A and 2B employ both isolation and cherry-picking strategies; andas noted in relation to FIGS. 2A and 2B, at least the gRNA (and also, insome embodiments, the nuclease) may be under control of an induciblepromoters thus employing induction in addition to isolation and cherrypicking. When induction is employed, clonal colonies of isolated cellsare allowed to grow for several to many doublings before editing isinduced to give the cells to be edited a chance to establish a colonybefore enduring the toxic effects of editing.

In an alternative embodiment, FIG. 2B also depicts replica plating 223of plate 208; however, only the small colonies 212 are picked and platedon plate 210. Selective replica plating of only select, small coloniesis accomplished by, in one embodiment, monitoring the growth of colonies212 and 214 on plate 208 (such as, e.g., by the automated JoVE ScanLag™device (Cambridge, Mass.)) and communicating the coordinates of thesmall colonies 212 to a 3D printer where a specialized replicator isfabricated (printed) and then used to transfer only the selectedcolonies (in this case the small colonies 212) to plate 210. Threedimensional printers are widely-available off-the-shelf from, e.g.,Makergear (Beachwood, Ohio); DigiKey Electronics (Minnesota); 3D Systems(Rock Hill, S.C.); EnvisionTEC (Dearborn, Mich.); ExOne (St.Clairsville, Ohio); and StrataSys (Eden Prairie, Ill.). See Examples 1-3below for methods and materials that may be used in this embodiment.

FIG. 2C shows the growth profiles of randomly-picked colonies oftransformed cells where the gRNA is under transcriptional control of aninducible promoter. Cells were picked from an agar plate and grown up inselective medium (selecting for both the engine and editing vectors)overnight in a 96-well microtiter plate format. An aliquot of the wellcontent of a parent microtiter plate (e.g., cell hotel or repository)was then transferred to two replica daughter microtiter plates, forexample using an automatic replicator such as the QRep replicators fromMolecular Devices (San Jose, Calif.) or the pin replicators from PhenixResearch Products (Chandler, N.C.). One microtiter plate received noinduction (top), and the other microtiter plate received gRNA inductionvia the pL inducible promoter for 1 hour at 42° C. (bottom). The wellmaps show the relative OD at 6 hours; the full growth curves are shownfor reference. The replica wells represent growth observed from the samecassette design with or without gRNA induction. While the majority ofthe wells for the no-induction plate show varying but normal growthprofiles, the induced plate shows that a large fraction of the gRNAdesigns is still active when induced, indicated by a large lag phase(very slow cell growth indicated by filled-in squares) before the cellsreach exponential growth. That is, the actively-editing cells havereduced viability due to DNA damage such that many cells in the coloniesdie off, and those edited cells that do survive may grow slowly to beginwith as the cellular machinery works to repair the edit and in any casethe edited colonies take some amount of time to “catch up” with theirunedited counterparts. Thus, FIG. 2C depicts an embodiment of editedcell selection using induction, solid-wall wells, isolation, andtargeted, super-Poisson loading.

Note that in this embodiment, importantly, putative lethal edits can beidentified. For example, if in the uninduced plate there is cell growth,but after induction of editing there is no growth (that is, not evenlagging growth), it is possible that the particular edit was lethal tothe cell. This concept can be tested by going back to the uninducedplate (e.g., the cell hotel or cell repository) and an aliquot of thecell colony corresponding to the putative lethal edit can be retrievedand tested.

Further, though the embodiment workflow depicted in FIG. 2C describesuse of 96-well plates, smaller or larger well plates can be used. It iscontemplated that for induced multiplex editing (that is, where two toseveral edits are made to cells simultaneously), larger-well plates areneeded to allow large initial colony formation (outgrowth) beforeediting. Again, because the double-strand cuts caused by active editingare largely toxic to the cells if not repaired, initial outgrowth of thecell colonies before editing is required—and in this case, the moreedits initiated per cell, the more toxic the editing process will be. Asdescribed above, the toxicity causes both cell death in the editedcolonies as well as a lag in growth for the edited cell colonies as thecells that do survive must repair and recover and catch up to thecolonies of unedited cells following editing. Thus, allowing for agreater outgrowth of cells before initiating a multiplex editing processincreases the odds of survival of multiplex-edited cells. This sameprinciple can be applied to cell colonies grown on agar plates (as shownin FIGS. 2A and 2B) or in 3D agarose space (as shown in FIGS. 2H and2I), where the colonies are plated less densely, and the colonies areallowed to grow to a bigger size. Similarly, if wells are used (as shownin FIGS. 2J and 2K), the wells are bigger to support larger cell colonygrowth; and if features are used to support cell growth (as shown inFIGS. 2D and 2E), the features are made larger to support growth oflarger colonies. See Example 2 below for methods and materials fordetermining OD in plates according to this embodiment.

FIG. 2D depicts yet another workflow 228 for screening and, optionally,selecting edited cells. In FIG. 2D, a substrate 230, such as, e.g., apolystyrene or glass substrate is prepared for deposition offunctionalized islands on which to grow cells. If the substrate isglass, the glass surface is prepared by oxidation, followed bydeposition of polystyrene 231, where the substrate 232 is then oxidizedagain to render the polystyrene temporarily hydrophobic. If thesubstrate 230 is polystyrene, it need only be oxidized 231 to render thepolystyrene hydrophobic. Once hydrophobic, collagen islands or patchesare printed 233 on the hydrophobic substrate. In certain embodiments,the collagen printed may be ordinary, non-crosslinked collagen orcrosslinked collagen, such as methylacrylated type-I collagen. Collagenis used routinely for cell culture, including as a matrix for growingcells for transplantation. Non-crosslinked collagen deposed on ahydrophobic substrate is stable at room temperature up to five months.

The collagen may be deposited by biologically-applied deposition andassembly devices and systems known in the art, including systems thatperform direct writing, microstamping, photolithography,electroprinting, extrusion, and inkjet deposition. For example, see thesystem sold commercially by Arrayjet (Roslin, UK). See also Bishop, etal., Genes & Diseases, 4(4):185-95 (2017); Zheng, et al., Anal.Biochem., 410(2):171-76 (2011); and Saunders and Derby, InternationalMaterials Review, 59(8):430-48 (2014). Regardless of the device used,the collagen is printed to form “islands” 236 on hydrophobic substrate234, then cells are flowed 235 across substrate 236 at a dilution wherethere is a Poisson distribution of cells on the islands (that is, suchthat each island has one or no cells, and the likelihood that any oneisland has more than one cell is low). Flowing the cells over substrate234 results in islands of collagen with one 238 or no cell attached. Aclose up 237 of substrate 234 with collagen islands with one cellattached is shown. Once the cells are loaded onto the collagen islandsor patches, the substrate is submerged in growth medium and the cellsare allowed to proliferate into normalized colonies 240. Because theareas between the islands are hydrophobic and not functionalized with asurface upon which cells can grow, the cells are isolated (e.g.,isolated) on substrate 234.

In this embodiment, the cells can be grown to terminal (normalized)growth such that the cells that have been edited and the cells that havenot been edited ultimately grow to equivalent sizes. Once the cellcolonies have been grown to terminal size, the cells can be pooled forfurther research or editing. As with other embodiments, this methodenriches (screens) for edited cells by eliminating the bias fromnon-editing cells and fitness effects from editing; that is, isolationalone enriches for editing cells such that all isolated colonies—notonly the slow-growing colonies—may be pooled into an “enriched” editedpool of cells. Alternatively, in this embodiment and particularly inbacterial systems, at least the gRNA is under the control of aninducible promoter. In this instance, isolation is carried out in thesame manner (e.g., here, Poisson loading of the collagen islands);however, instead of growing the cells to colonies of terminal size, thecells are allowed to double to approximately between 2 and 200, orbetween 5 and 150, or between 10 and 100 times to form clonal colonies,then editing is induced by heating the substrate (e.g., fortemperature-induced editing) or flowing chemicals over the substrate(e.g., sugars, antibiotics for compound-induced editing). Afterinduction of editing, the cells are allowed to grow to continue toestablish colonies, and the growth of the colonies can be normalized andthe cells pooled or cell growth is monitored such that slow growingcolonies can be identified and selected.

FIG. 2E depicts an exemplary substrate and workflow 2010 for isolatingand selecting cells using collagen pillars isolated from one another bypolyethylene glycol (PEG). In a first step, a substrate 2012 (glass orplastic) is coated with a layer of chromium, and a photolithographicmask is used to expose a pattern in the chromium 2013. Substrate 2014thus comprises a chromium mask with features 2016 (circular areas) wherethe collagen will be deposited. The size of the features may vary withthe type of cells that are to be isolated and cultured. For example, ifbacterial cells are being cultured, the features may be on the order of2-5 μm or larger, if yeast cells are being cultured, the features may beon the order of 3-8 μm or larger, and if mammalian cells are beingcultured, the features may be on the order of 100-300 μm or larger. Thechromium mask is deposited at a depth appropriate for the height of thecollagen pillars. At step 2015, collagen is deposited as features 2020on substrate 2018. In some aspects, the collagen is photo-crosslinkablecollagen, such as methacrylated type-I collagen (CMA).

At step 2017, the chromium mask is removed leaving only the pillars ofcollagen 2020 on substrate 2022. At step 2019, polyethylene glycol (PEG)is deposited between the pillars on substrate 2024, forming an isolatingbarrier between the pillars 2020. At step 2021, a close up of substrate2024 in a top view and side view depicts a substrate 2030 where cells inan appropriate dilution are flowed over the substrate 2030 such thatsome pillars 2026 will have a cell 2032 deposited on them and somepillars 2028 will not. Finally, in step 2023 the cells are allowed togrow and to populate the collagen pillars, growing down and into thepillars. In some pillars 2036, the cells populate the pillars quickly,whereas on other pillars the cells populate more slowly. Also seen arepillars where cells were not deposited 2028.

In some aspects of this embodiment, the cell colonies are grown toterminal size and then all cells are harvested. As described above,because isolation eliminates the bias of non-editing cells and fitnesseffects from editing, isolation alone enriches for editing cells suchthat all isolated colonies—not only the slow-growing colonies—may bepooled into an “enriched” edited pool of cells. In this aspect, editingdoes not need to be induced. Yet in another aspect of this embodiment,the rate of cell growth can be monitored—for example fast growingcolonies 2036 can be distinguished from slow growing colonies 2034—andthe slow growing colonies can be selected for further study or anotherround of editing. Further, it should be clear to one of ordinary skillin the art given the discussion herein that other methods and materialsmay be used to fabricate pillars or wells on a substrate, where collagenmay be deposited to grow colonies of isolated cells. Thus, the workflowdepicted in FIG. 2E employs isolation achieved by Poisson loading ontofeatures isolated from one another by PEG (or other compound that doesnot allow cell growth). In this embodiment, isolation and normalizationalone may be employed, or isolation and cherry picking may be employed.

FIGS. 2F and 2G depict workflows for identifying edited cells afternucleic acid-guided nuclease genome editing where cells are isolatedinto droplets in an emulsion and may then be selected (e.g., cherrypicked). Cells are arrayed and sorted dynamically in droplets in a flowstream. A stream of an aqueous solution (cells in a cell growth medium)are introduced into a stream of carrier fluid, such as a non-polarsolvent or oil, where droplets of the aqueous solution are formed. Theconcentration of the cells should be dilute enough that most of thedroplets contain no more than a single cell with only a smallstatistical chance that a droplet will contain two or more cells.Dilution is effected to ensure that for the large majority ofmeasurements, the level of reporter (colorimetric reporter or cellulardensity) corresponds to a single, starting cell in a droplet.

The flow stream in the main channel is typically, but not necessarily,continuous and may, in some embodiments, be stopped and started,reversed or change speed. The pressure of the flows of the carrier fluidand the aqueous solution—as well as the pressure at the dropletgeneration region—can be regulated by, e.g., adjusting the pressure onthe carrier and aqueous fluid reservoirs. By controlling the pressuredifference between the oil and aqueous sources at the droplet generationregion, the size and periodicity of the droplets generated may beregulated. Alternatively, one or more valves may be coincident to eitherthe droplet generation region or the aqueous feed to control the flow ofsolution. The fluids used in the exemplary modules depicted in FIGS. 2Fand 2G may contain additives such as surfactants that reduce surfacetension. Exemplary surfactants include Tween, Span, fluorinated oils,and other agents that are soluble in oil relative to water.

In FIG. 2F, a workflow 260 isolates cells into droplets, which are thenarrayed in wells. First, a stream of an emulsifier such as a non-polarsolvent (e.g., decane) or oil is flowed from reservoir 262 toward adroplet generator 266 (e.g., a T-junction, cross-junction, or flowfocusing device) where the flow of the emulsifier meets the flow oftransformed cells in an aqueous medium from a reservoir 264. In anautomated multi-module cell processing instrument, the transformed cellsmay have been transferred to reservoir 264 from a transformation module(e.g., a flow-through electroporation device) as described in relationto FIGS. 3A-3D below. At the junction in the droplet generator 266 wherethe flow of the nonpolar solvent and the flow of the cells in aqueousmedium meet, droplets 268 are formed. The concentration of thetransformed cells in the aqueous medium, again, is controlled so eachdroplet comprises an average of one-half cell or less, such that themajority of droplets comprises either one cell or no cells. Droplets 268each comprise a cell 270, and droplets 269 comprise no cells. Thedroplets proceed through a conduit until they are dispensed, one at atime, into a substrate with wells 272 containing medium. Each well inthe substrate should comprise a single droplet, some droplets 268containing a cell, and some droplets 269 without a cell.

If the editing system transformed into the cells is not an induciblesystem, editing may begin as soon as the necessary nucleic acid-guidednuclease editing components are delivered to and transcribed—and in somecases, translated—in the cell. That is, the editing process commences ator shortly after transformation. Alternatively, once transformationtakes place, the transformed cells in reservoir 264 (and proceedingthrough the droplet generation device) may be cooled to prevent or slowthe initiation of editing until the cells are isolated or until thecells are deposited into a well in substrate 272. The isolated cells canbe grown to establish colonies, and this growth can be measured, e.g.,by a spectrophotometer (not shown).

Alternatively, one or more components of the nucleic acid-guidednuclease editing system (e.g., at least the gRNA and in some embodimentsthe nuclease as well) may be under the control of an inducible promoter.Thus, in one embodiment, the wells of the substrate may comprise, inaddition to medium, a compound that activates an inducible promoterdriving the gRNA and/or nuclease, such that editing of the cellulargenome is not initiated until the cell droplets are deposited into awell in the substrate 272. In yet another alternative, one or morecomponents of the nucleic acid-guided nuclease editing system (e.g., atleast the gRNA and also the nuclease) may be under the control of aninducible promoter where activity of the promoter is induced by anincrease in temperature. In this instance, instead of an inducingcompound in the medium in the wells, the substrate with the wells 272 isheated to an appropriate temperature to activate the induciblepromoter(s). In this embodiment, the isolated cells may be grown for aperiod of time (e.g., 2-200 doublings) before the inducible promotersare activated. Once activated, editing commences for a period of time,then the edited cells are allowed to grow into colonies where the growthof the colonies is monitored by, e.g., the automated cell colony sizemonitors described above and marketed by IncuCyte (Ann Arbor, Mich.).

Further, arraying of cells in single-cell isolation as contemplated bythe workflow as described in relation to FIG. 2F may be performed with“off the shelf” instrumentation, such as that sold by CellenONE (Lyon,France); Cytena (Freiberg, Germany); Cell Microsystems (the Cellraft™technology, Research Triangle Park, N.C.); Galt, Inc. (San Carlos,Calif.); and 10× Genomics (Pleasanton, Calif.). (See also Zhang, et al.,Scientific Reports, 7:41192 (2017).) Thus, the workflow depicted in FIG.2F employs both “liquid walls” and “solid walls” for isolation and maybe used to isolate and grow the isolated cells to colonies of terminalsize (enrich) only, or, optionally, editing may be induced and cellgrowth monitored so that selection (e.g., cherry picking) may beemployed.

FIG. 2G depicts another module 280 for identifying edited cells afternucleic acid-guided nuclease genome editing where cells are isolatedinto droplets. In FIG. 2G like FIG. 2F, a stream of an emulsifier suchas a non-polar solvent (e.g., decane) or oil is flowed from reservoir262 toward a droplet generator 266 (e.g., a T-junction, cross-junction,or flow focusing device) where the flow of the emulsifier meets the flowof transformed cells in an aqueous medium that originated in reservoir264. At the junction in the droplet generator 266 where the flow of thenonpolar solvent and the flow of the cells in aqueous medium meet,aqueous droplets 268 are formed and transported in the nonpolar solvent(carrier phase). The concentration of the transformed cells in theaqueous medium is controlled such that an average of one-half cell iscontained in each droplet; thus, the majority of droplets compriseseither one cell (e.g., 268) or no cells (e.g., 269). The dropletsproceed through a conduit until they reach an induction module 274.

An induction module is used if transcription of one or more componentsof the nucleic acid-guided nuclease editing system (e.g., at least thegRNA) is inducible. For example, if one or more components of thenucleic acid-guided nuclease editing system (e.g., one or both of thegRNA and nuclease) is under the control of an inducible promoter that isinduced by a chemical compound (e.g., arabinose, rhamnose), theinduction module may dispense the induction compound into the droplet,by, e.g., droplet merger. (See, e.g., U.S. Pat. No. 9,347,059 toSaxonov; US Pub. No. 2011/0053798 to Hindson, et al.; and US Pub. No.2008/0014589 to Link, et al.)

Alternatively, at least the gRNA is under the control of an induciblepromoter where activity of the promoter is induced by an increase intemperature, such as a pL promoter system. In this instance, instead ofadding an inducing compound, the temperature of the droplets in theinduction module is raised to an appropriate temperature to activate theinducible promoter(s). In cells with active gRNAs 276 editing isinduced. In cells with inactive gRNAs (or other editing machinery thatis inactive) 277, editing does not take place.

Once editing is initiated in the droplets, the droplets are may betransported to a growth module 281 which can maintain the droplets atthe higher temperature initially to continue the editing process. Oncetime for editing passes, the temperature is reduced to allow the cellsto grow into colonies. Colonies of edited cells, which grow slowly inrelation to colonies of unedited cells, are shown at 278, and uneditedcells, which grow more quickly, are shown at 282. After growth in growthmodule 281, the droplets containing the cells can be transported to adetector 284, where, e.g., droplets with densely grown cell colonies 282(unedited cells) are distinguished from droplets with less densely growncell colonies 278 (edited cells). Once each droplet is assessed forgrowth, the droplets proceed to the cell sorter 286 where the cells withdensely grown cell colonies 282 are shuttled into a conduit leading toreservoir 288 and cells with less densely grown colonies 278 areshuttled into a conduit leading to reservoir 287. The detector measurescell growth by, e.g., a spectrophotometer (not shown) to detect opticaldensity or the readout of a colorimetric assay. In some embodiments, thedetector 284 and cell sorter 286 may be combined into a singleinstrument.

The detector can be any device or method for interrogating a cell as itpasses through a detection region. Typically, cells (or the droplets inwhich the cells are contained) are sorted according to one or morepredetermined characteristics that are directly or indirectlydetectable, and the detector is adapted to detect the characteristic.One detector of particular use in the modules shown in FIGS. 2G and 2His an optical detector, such as a microscope or spectrophotometer, whichmay be coupled to a computer and/or other image processing orenhancement device, to process images. Cells can be sorted by theoptical density of cells within the droplet, or by intensity of acolorimetric assay that correlates to cell growth, such as the cellproliferation assays described above. There is no limit to the kindand/or number of cellular characteristics that may be identified ormeasured as long as the readout for the characteristic(s) can besufficiently identified and detected to distinguish—e.g., as a proxy forgrowth of—putatively edited cells from putative nonedited cells.

The cells are analyzed and sorted based on the intensity of a signaldetected as the droplets pass through a detection region or window. Thesignal may be collected by a microscope or spectrophotometer andmeasured by a photo multiplier tube. A computer digitizes the signal andcontrols the flow via, e.g., valve action or electroosmotic potential.Cells having a level of reporter (or OD) below a selected threshold orwithin a selected range are diverted into a predetermined outlet orreservoir. For example, a cell-sorting device may comprise aspectrophotometer where the optical density of each cell is read as itpasses by the light beam. The optical signal is collected and projectedonto a cathode of a photomultiplier tube. Optionally, part of the lightmay be directed onto a charge-coupled device (CCD) camera for imaging.As the cells pass by the spectrophotometer detection window, the cellsare directed to conduits that lead to the reservoirs that collect editedcells and non-edited cells or empty droplets depending onvoltage-potential settings. The voltages on the electrodes are providedby a pair of amplifiers powered by a power supply. The photomultipliertube signal is digitized by a processor, which also controls the highvoltage settings. In addition to optical density or colorimetric assays,cell colonies may be sorted by density; for example, see Nam, et al.,BioMicrofluidics, 6:024120 (2012); and Norouzi, et al., PLoS One,12(7):e0180520 (2017). Additionally, there are commercial single cellsorting devices available, for example from nanocellect Biomedical, Inc.(San Diego, Calif.); NanoCell Inc. (Mountain View, Calif.); and SiliconBiosystems (the DEParray™ technology, Castel, Italy).

FIG. 2H is an exemplary workflow 2100 a for optimizing the observedpresence of edited cells after nucleic acid-guided nuclease genomeediting that may be performed in an automatedisolation/growth/editing/normalization module, and, optionally, as partof an automated multi-module cell editing instrument. First, transformedcells 2104 are suspended at a pre-determined density in medium plusalginate (solidifying agent) in a vessel 2102 containing, optionally,antibiotics or other selective compounds to allow only cells that havebeen transformed with both the engine vector and editing vector (if twovectors are used) or a combined engine/editing vector to grow. Again, insome embodiments two vectors, an engine vector and an editing vector,are used in some embodiments a single vector comprising all necessaryexogenous components for nucleic acid-guided nuclease editing is used.The medium used with depend, of course, on the type of cells beingedited—e.g., bacterial, yeast or mammalian. For example, medium forbacterial growth includes LB, SOC, M9 Minimal medium, and Magic medium;medium for yeast cell growth includes TPD, YPG, YPAD, and syntheticminimal medium; and medium for mammalian cell growth includes MEM, DMEM,IMDM, RPMI, and Hanks.

Natural polymers and proteins able to form hydrogels are alginate,chitosan, hyaluronan, dextran, collagen, and fibrin; synthetic examplesof synthetic polymers and proteins able to form hydrogels includepolyethylene glycol, poly(hydroxyethyl methacrylate, polyvinyl alcohol,and polycaprolactone. Alginate has been used as a preferred solidifyingagent in the methods described herein due to a number of advantageousproperties. Alginates are polysaccharides that consist of linear(unbranched) 1,4 linked residues of D-mannuronic acid and its C5-epimerD-guluronic acid. Alginates have a high affinity for alkaline earthmetals and ionic hydrogels can be formed in the presence of divalentcations except Mg+2. Chelation of the gel-forming ion occurs between twoconsecutive residues in the alginate chain, and an intermolecular gelnetwork is formed as a result of a cooperative binding of consecutiveresidues in different alginate chains. Advantageously, ionically-gelledalginate can be dissolved by treatment with chelating agents fordivalent cations such as citrate and ethylenediaminetetraacetic acid(EDTA) or hexametaphosphate. A 2% (w/v) alginate in medium was found toproperly isolate cells; however appropriate ranges for the percentage ofalginate in a growth medium include 0.25% to 6% (w/v) alginate, or 0.5%to 5% (w/v) alginate, or 1% to 4% (w/v) alginate, or 2% to 3% (w/v)alginate. In addition, neither of the processes of solidifying and ofre-liquefying the alginate/medium (described in more detail below)impact cell viability. Moreover, induction of editing by elevating thetemperature of the bulk gel to 42° C. (described in more detail below)does not impact the integrity of the solidified medium or thesegregation of the isolated clonal cell colonies.

The culture of mammalian cells using hydrogels has been performed tomimic the 3D cell environments found in tissue, allowing for morebiologically-relevant cellular environments. As with tissue mimetics, inthe context of mammalian cell editing alginates may be chemicallyfunctionalized to alter physiochemical and biological characteristicsand properties so as to better bind and promote the growth of mammaliancells once the cells have been isolated in the solidified alginatemedium. As cells do not have receptors that recognize alginate,proliferation and differentiation of some mammalian cells within analginate hydrogen require signaling molecules and matrix interaction.For example, cell attachment peptides, especially the sequence RGD(arginine-glycine-aspartic acid), have been shown to improve cellularadaptability to matricies, as is the case with alginate. Using aqueouscarbodiimide chemistry, alginate can be modified by covalently graftingpeptide sequence to the alginate molecule. (For a comprehensivediscussion of 3D cell culture in alginate hydrogels, see Andersen, etal., Microarrays, 4:133-61 (2015).) Alternatively, as described above,mammalian cells can be grown on beads where the beads are then suspendedin the alginate medium.

Once the cells are suspended at an appropriate density, the alginate inthe medium is solidified 2101 by, e.g., addition of CaCl₂ (describedbelow in relation to Example 7). Note that some areas of the solidifiedalginate have no cells 2106 and some areas have one cell 2104. Next, thecells are allowed to grow 2103 for a pre-determined approximate numberof doublings. Because the cells are fixed in three-dimensional space,the resulting colonies 2108 are fixed in three-dimensional space. Thecolonies are grown to terminal size 2107 (that is, edited and non-editedcell colony growth is normalized), sodium citrate is added 209 to themedium such that the solidified medium/alginate re-liquifies and thecells from the colonies 2114, 2116 (comprising to edited and uneditedcells, respectively) are suspended in liquid medium once again. Once themedium is re-liquified, the cells are recovered and subjected toanalysis 2111 or are used in a second round of editing 2113. Again,because the combination of the processes of isolation and normalizationovercomes growth bias from unedited cells or cells exhibiting fitnesseffects as the result of edits made, the combination of the processes ofisolation and normalization alone enriches the total population of cellswith cells that have been edited; that is, isolation and normalization(e.g., growing colonies to terminal size) allows for high-throughputenrichment of edited cells.

FIG. 2I is yet another exemplary workflow 2100 for optimizing theobserved presence of edited cells after nucleic acid-guided nucleasegenome editing that may be performed in an automatedisolation/growth/editing/normalization module, and, optionally, as partof an automated multi-module cell editing instrument. FIG. 2I, unlikeFIG. 2H, employs induction of transcription of the gRNA. First,transformed cells 2104 are suspended at a pre-determined density inmedium plus alginate (solidifying agent) in a vessel 2102 containing,optionally, antibiotics or other selective compounds to allow only cellsthat have been transformed with both the engine vector and editingvector (if two vectors are used) or a combined engine/editing vector togrow. Again, in some embodiments a single vector comprising allnecessary exogenous components for nucleic acid-guided nuclease editingis used. As described above, the medium used with depend, of course, onthe type of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks.

Once the cells 2104 are suspended at an appropriate density, thealginate in the medium is solidified 2101 by, e.g., addition of CaCl₂(described below in relation to Example 7). Next, the cells are allowedto grow for a pre-determined approximate number of doublings 2103.Because the cells are fixed in three-dimensional space, the resultingcolonies are fixed in three-dimensional space. After a number ofdoublings, editing is induced 2105 and some of the cells in the colonieswhere editing takes place die as a result of double-stranded breaks inthe genome that are not repaired. Once editing has taken place, thecolonies are allowed to grow to terminal size 2107. Once the coloniesgrow to terminal size (that is, edited and non-edited cell colony growthis normalized), sodium citrate 2109 is added to the medium such that thesolidified medium/alginate re-liquifies and the cells from the coloniesare suspended in liquid medium once again. Because the combination ofthe processes of isolation and normalization overcomes growth bias fromunedited cells or cells exhibiting fitness effects as the result ofedits made, the combination of the processes of isolation andnormalization alone enriches the total population of cells with cellsthat have been edited; that is, isolation, optional induction, andnormalization (e.g., growing colonies to terminal size) allows forhigh-throughput enrichment of edited cells.

FIG. 2J depicts a solid wall device 2250 and a workflow for isolatingcells in microwells in the solid wall device, where in this exemplaryworkflow at least the gRNA is optionally be under the control of aninducible promoter, particularly if editing in bacterial cells. At thetop left of the FIG. (i), there is depicted solid wall device 2250 withmicrowells 2252. A section 2254 of substrate 2250 is shown at (ii), alsodepicting microwells 2252. At (iii), a side cross-section of solid walldevice 2250 is shown, and microwells 2252 have been loaded, where, inthis embodiment, Poisson or substantial Poisson loading has taken place;that is, each microwell has one or no cells, and the likelihood that anyone microwell has more than one cell is low. Note wells 2256 have onecell loaded. At (iv), workflow 2240 is illustrated where substrate 2250having microwells 2252 shows microwells 2256 with one cell permicrowell, microwells 2257 with no cells in the microwells, and onemicrowell 2260 with two cells in the microwell. In step 2251, the cellsin the microwells are allowed to double approximately 2-150 times toform clonal colonies (v), then editing optionally is induced 2253 byheating the substrate (e.g., for temperature-induced editing) or flowingchemicals under or over the substrate (e.g., sugars, antibiotics forchemical-induced editing) or by moving the solid wall device to adifferent medium, particularly facile if the solid wall device is placedon a membrane which forms the bottom of microwells 2252 (membrane notshown). Induction of editing is optional, however. If editing is notinduced, editing begins, e.g., upon transformation of the editing“machinery” into the cell or shortly thereafter.

After optional induction of editing 2253, many cells in the colonies ofcells that have been edited die as a result of the double-strand cutscaused by active editing and there is a lag in growth for the editedcells that do survive but must repair and recover following editing(microwells 2258), where cells that do not undergo editing thrive(microwells 2259) (vi). All cells are allowed to continue grow toestablish colonies and normalize, where the colonies of edited cells inmicrowells 2258 catch up in size and/or cell number with the cells inmicrowells 2259 that do not undergo editing (vii). Once the cellcolonies are normalized, either pooling 2260 of all cells in themicrowells can take place, in which case the cells are enriched foredited cells by eliminating the bias from non-editing cells and fitnesseffects from editing; alternatively, colony growth in the microwells ismonitored after editing, and slow growing colonies (e.g., the cells inmicrowells 2258) are identified and selected 2261 (e.g., “cherrypicked”) resulting in even greater enrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks. For culture ofadherent cells, cells may be disposed on beads or another type ofscaffold suspended in medium. Most normal mammalian tissue-derivedcells—except those derived from the hematopoietic system—are anchoragedependent and need a surface or cell culture support for normalproliferation. In the rotating growth vial described herein,microcarrier technology is leveraged. Microcarriers of particular usetypically have a diameter of 100-300 μm and have a density slightlygreater than that of the culture medium (thus facilitating an easyseparation of cells and medium for, e.g., medium exchange) yet thedensity must also be sufficiently low to allow complete suspension ofthe carriers at a minimum stirring rate in order to avoid hydrodynamicdamage to the cells. Many different types of microcarriers areavailable, and different microcarriers are optimized for different typesof cells. There are positively charged carriers, such as Cytodex 1(dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-AldrichLabware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170(polystyrene-based); collagen or ECM (extracellular matrix)-coatedcarriers, such as Cytodex 3 (dextran-based, GE Healthcare) or HyQ-spherePro-F 102-4 (polystyrene-based, Thermo Scientific); non-chargedcarriers, like HyQspheres P 102-4 (Thermo Scientific); or macroporouscarriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose(Cytopore, GE Healthcare).

The solid wall devices can provide populations of cells with varyingedits and/or percentages of clonality. It has been determined thatflowing medium over the retentate side surface of the SWIIN, e.g., atangential or sheer flow across the top of the perforate member of theSWIIN, will flush off the tops (“muffin tops”, see FIG. 9D) of the cellcolonies over-growing the wells of the SWIIN without contaminating thewells containing other cells; i.e., depositing flushed cells in wells.In one embodiment, cells are allowed to grow to a desired state, forexample when some of the colonies—fast-growing colonies, which arelikely to be unedited cells—have over-grown the wells, then the “muffintops” are flushed off. In a next round, the cells again are allowed togrow again to a desired state, for example when some or many more of thecolonies have over-grown the wells, then the “muffin tops” are flushedoff, and additional rounds of cell growth and flushing/collection cancontinue as desired. After a desired number of rounds of cell growth andcollection, 1) the cells can be collected and pooled, 2) certaincollections may be discarded (such as the first collection of cellswhich are more likely to be unedited cells) and the rest of thecollections pooled, or 3) each round of collection may be kept separateand analyzed for clonality, percentage of edited cells, etc. Thisembodiment requires monitoring of cell growth by, e.g., imaging, asdescribed in relation to FIG. 2G or 5I.

FIG. 2K depicts a solid wall device 2250 and a workflow forsubstantially isolating cells in microwells in a solid wall device,where in this workflow—as in the workflow depicted in FIG. 2J—optionallyat least the gRNA and nuclease is under the control of an induciblepromoter, particularly in bacterial systems. At the top left of the FIG.(i), there is depicted a solid wall device 2250 with microwells 2252. Asection 2254 of substrate 2250 is shown at (ii), also depictingmicrowells 2252. At (iii), a side cross-section of solid wall device2250 is shown, and microwells 2252 have been loaded, where, in thisembodiment, substantial Poisson loading has taken place; that is, onemicrowell 2257 has no cells, and some microwells 2276, 2278 have a fewcells. In FIG. 2K, cells with active gRNAs are shown as solid circles,and cells with inactive gRNAs are shown as open circles. At (iv)workflow 2270 is illustrated where substrate 2250 having microwells 2252shows three microwells 2276 with several cells all with active gRNAs,microwell 2257 with no cells, and two microwells 378 with some cellshaving active gRNAs and some cells having inactive gRNAs. In step 2271,the cells in the microwells are allowed to double approximately 2-150times to form clonal colonies (v), then editing optionally is induced2273 by heating the substrate (e.g., for temperature-induced editing) orflowing chemicals under or over the substrate (e.g., sugars, antibioticsfor chemical-induced editing) or by moving the solid wall device to adifferent medium, particularly facile if the solid wall device is placedon a membrane which forms the bottom of microwells 2252. Again, editingneed not be inducible, in which case editing commences at or shortlyafter transformation and is likely taking place as the cells are beingdeposited in the wells.

After editing 2273, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 2278),where cells that do not undergo editing thrive (microwells 2279) (vi).Thus, in microwells 2278 where only cells with active gRNAs reside(cells depicted by solid circles), most cells die off; however, inmicrowells 2279 containing cells with inactive gRNAs (cells depicted byopen circles), cells continue to grow and are not impacted by activeediting. The cells in each microwell (2278 and 2279) are allowed to growto continue to establish colonies and normalize, where the colonies ofedited cells in microwells 2278 catch up in size and/or cell number withthe unedited cells in microwells 2279 that do not undergo editing (vii).Note that in this workflow 2270, the colonies of cells in the microwellsare not clonal; that is, not all cells in a well arise from a singlecell. Instead, the cell colonies in the well may be mixed colonies,arising in many wells from two to several different cells. Once the cellcolonies are normalized, either pooling 2290 of all cells in themicrowells can take place, in which case the cells are enriched foredited cells by eliminating the bias from non-editing cells and fitnesseffects from editing or cells may be flushed from the SWIIN andcollected at various time points; alternatively, colony growth in themicrowells is monitored after editing, and slow growing colonies (e.g.,the cells in microwells 2278) are identified and selected 2291 (e.g.,“cherry picked”) resulting in even greater enrichment of edited cells.

Automated Instruments Comprising Screening and Selection Modules

FIG. 3A depicts an exemplary automated multi-module cell processinginstrument 300 comprising a cell enrichment/selection module 340 to,e.g., perform one of the exemplary workflows described above, as well asadditional exemplary modules. Illustrated is a gantry 302, providing anautomated mechanical motion system (actuator) (not shown) that suppliesXYZ axis motion control to, e.g., modules of the automated multi-modulecell processing instrument 300, including, e.g., a liquid handlingsystem 358 with an air displacement pipette 332. In some automatedmulti-module cell processing instruments, the air displacement pipettoris moved by a gantry and the various modules and reagent cartridgesremain stationary; however, in other embodiments, the pipetting systemmay stay stationary while the various modules are moved. Also includedin the automated multi-module cell processing instrument 300 is wash orreagent cartridge 304, comprising reservoirs 306. As described below inrespect to FIG. 3B, wash or reagent cartridge 304 may be configured toaccommodate large tubes, for example, wash solutions, or solutions thatare used often throughout an iterative process. In one example, wash orreagent cartridge 304 may be configured to remain in place when two ormore reagent cartridges 310 are sequentially used and replaced. Althoughreagent cartridge 310 and wash or reagent cartridge 304 are shown inFIG. 3A as separate cartridges, the contents of wash cartridge 304 maybe incorporated into reagent cartridge 310.

The exemplary automated multi-module cell processing instrument 300 ofFIG. 3A further comprises a cell growth module 334. In the embodimentillustrated in FIG. 3A, the cell growth module 334 comprises two cellgrowth vials 318, 320 (described in greater detail below with relationto FIG. 3E) as well as a cell concentration module 322. In alternativeembodiments, the cell concentration module 322 may be separate from cellgrowth module 334, e.g., in a separate, dedicated module. Alsoillustrated as part of the automated multi-module cell processinginstrument 300 of FIG. 3A is enrichment/selection module 340, served by,e.g., air displacement pipettor 332. Also seen are a waste repository326, and a nucleic acid assembly/desalting module 314 comprising areaction chamber or tube receptacle (not shown) and further comprising amagnet 316 to allow for purification of nucleic acids using, e.g.,magnetic solid phase reversible immobilization (SPRI) beads (AppliedBiological Materials Inc., Richmond, BC). The reagent cartridge,transformation module, and cell growth module are described in greaterdetail below. For additional information regarding integrated automatedmulti-module cell processing systems see U.S. Ser. No. 10,253,316,issued 9 Apr. 2019; U.S. Pat. No. 10,329,559, issued 25 Jun. 2019; andU.S. Pat. No. 10,323,242, issued 18 Jun. 2019; and U.S. Ser. No.16/412,175, filed 14 May 2019; Ser. No. 16/412,195, filed 14 Jun. 2019;and Ser. No. 16/423,289, filed 28 May 2019.

FIG. 3B depicts an exemplary combination reagent cartridge andelectroporation device 310 (“cartridge”) that may be used in anautomated multi-module cell processing instrument along with thescreening/selection module. In certain embodiments the material used tofabricate the cartridge is thermally-conductive, as in certainembodiments the cartridge 310 contacts a thermal device (not shown),such as a Peltier device or thermoelectric cooler, that heats or coolsreagents in the reagent receptacles or reservoirs 312. Reagentreceptacles or reservoirs 312 may be receptacles into which individualtubes of reagents are inserted as shown in FIG. 3B, or the reagentreceptacles may hold the reagents without inserted tubes. Additionally,the receptacles in a reagent cartridge may be configured for anycombination of tubes, co-joined tubes, and direct-fill of reagents.

In one embodiment, the reagent receptacles or reservoirs 312 of reagentcartridge 310 are configured to hold various size tubes, including,e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorfor microcentrifuge tubes. In yet another embodiment, all receptacles maybe configured to hold the same size tube, e.g., 5 ml tubes, andreservoir inserts may be used to accommodate smaller tubes in thereagent reservoir (not shown). In yet another embodiment—particularly inan embodiment where the reagent cartridge is disposable—the reagentreservoirs hold reagents without inserted tubes. In this disposableembodiment, the reagent cartridge may be part of a kit, where thereagent cartridge is pre-filled with reagents and the receptacles orreservoirs sealed with, e.g., foil, heat seal acrylic or the like andpresented to a consumer where the reagent cartridge can then be used inan automated multi-module cell processing instrument. As one skilled inthe art will appreciate given the present disclosure, the reagentscontained in the reagent cartridge will vary depending on workflow; thatis, the reagents will vary depending on the processes to which the cellsare subjected in the automated multi-module cell processing instrument.

Reagents such as cell samples, enzymes, buffers, nucleic acid vectors,expression cassettes, proteins or peptides, reaction components (suchas, e.g., MgCl₂, dNTPs, nucleic acid assembly reagents, gap repairreagents, and the like), wash solutions, ethanol, and magnetic beads fornucleic acid purification and isolation, etc. may be positioned in thereagent cartridge at a known position. In some embodiments of cartridge310, the cartridge comprises a script (not shown) readable by aprocessor (not shown) for dispensing the reagents. Also, the cartridge310 as one component in an automated multi-module cell processinginstrument may comprise a script specifying two, three, four, five, tenor more processes to be performed by the automated multi-module cellprocessing instrument. In certain embodiments, the reagent cartridge isdisposable and is pre-packaged with reagents tailored to performingspecific cell processing protocols, e.g., genome editing or proteinproduction. Because the reagent cartridge contents vary whilecomponents/modules of the automated multi-module cell processinginstrument may not, the script associated with a particular reagentcartridge matches the reagents used and cell processes performed. Thus,e.g., reagent cartridges may be pre-packaged with reagents for genomeediting and a script that specifies the process steps for performinggenome editing in an automated multi-module cell processing instrument,or, e.g., reagents for protein expression and a script that specifiesthe process steps for performing protein expression in an automatedmulti-module cell processing instrument.

For example, the reagent cartridge may comprise a script to pipettecompetent cells from a reservoir, transfer the cells to a transformationmodule (such as flow through electroporation device 330 in reagentcartridge 310), pipette a nucleic acid solution comprising a vector withexpression cassette from another reservoir in the reagent cartridge,transfer the nucleic acid solution to the transformation module,initiate the transformation process for a specified time, then move thetransformed cells to yet another reservoir in the reagent cassette or toanother module such as a cell growth module in the automatedmulti-module cell processing instrument. In another example, the reagentcartridge may comprise a script to transfer a nucleic acid solutioncomprising a vector from a reservoir in the reagent cassette, nucleicacid solution comprising editing oligonucleotide cassettes in areservoir in the reagent cassette, and a nucleic acid assembly mix fromanother reservoir to the nucleic acid assembly/desalting module (314 ofFIG. 3A). The script may also specify process steps performed by othermodules in the automated multi-module cell processing instrument. Forexample, the script may specify that the nucleic acid assembly/desaltingreservoir be heated to 50° C. for 30 min to generate an assembledproduct; and desalting and resuspension of the assembled product viamagnetic bead-based nucleic acid purification involving a series ofpipette transfers and mixing of magnetic beads, ethanol wash, andbuffer. These processes are described in greater detail infra.

As described in relation to FIGS. 3C and 3D below, the exemplary reagentcartridges 310 for use in the automated multi-module cell processinginstruments may include one or more electroporation devices 330,preferably flow-through electroporation devices. Electroporation is awidely-used method for permeabilization of cell membranes that works bytemporarily generating pores in the cell membranes with electricalstimulation. Applications of electroporation include the delivery ofDNA, RNA, siRNA, peptides, proteins, antibodies, drugs or othersubstances to a variety of cells such as mammalian cells (includinghuman cells), plant cells, archea, yeasts, other eukaryotic cells,bacteria, and other cell types. Electrical stimulation may also be usedfor cell fusion in the production of hybridomas or other fused cells.During a typical electroporation procedure, cells are suspended in abuffer or medium that is favorable for cell survival. For bacterial cellelectroporation, low conductance mediums, such as water, glycerolsolutions and the like, are often used to reduce the heat production bytransient high current. In traditional electroporation devices, thecells and material to be electroporated into the cells (collectively“the cell sample”) are placed in a cuvette embedded with two flatelectrodes for electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength; however, the flow-through electroporation devices included inthe reagent cartridges such as those shown in FIGS. 3B-3D achieve highefficiency cell electroporation with low toxicity. The reagentcartridges of the disclosure allow for particularly easy integrationwith robotic liquid handling instrumentation that is typically used inautomated instruments and systems such as air displacement pipettors.Such automated instrumentation includes, but is not limited to,off-the-shelf automated liquid handling systems from Tecan (Mannedorf,Switzerland), Hamilton (Reno, Nev.), Beckman Coulter (Fort Collins,Colo.), etc. as described above.

FIGS. 3C and 3D are top perspective and bottom perspective views,respectively, of an exemplary flow-through electroporation device 350that may be part of reagent cartridge 300 in FIG. 3B or may be containedin a separate module (e.g., a transformation/transfection module). FIG.3C depicts a flow-through electroporation unit 350. The flow-throughelectroporation unit 350 has wells that define cell sample inlets 352and cell sample outlets 354. FIG. 3D is a bottom perspective view of theflow-through electroporation device 350 of FIG. 3C. An inlet well 352and an outlet well 354 can be seen in this view. Also seen in FIG. 3Dare the bottom of an inlet 362 corresponding to well 352, the bottom ofan outlet 364 corresponding to the outlet well 354, the bottom of adefined flow channel 366 and the bottom of two electrodes 368 on eitherside of flow channel 366. Additionally, flow-through electroporationdevices may comprise push-pull pneumatic means to allow multi-passelectroporation procedures; that is, cells to be electroporated may be“pulled” from the inlet toward the outlet for one pass ofelectroporation, then be “pushed” from the outlet end of theflow-through electroporation device toward the inlet end to pass betweenthe electrodes again for another pass of electroporation. Further, thisprocess may be repeated one to many times. Further, other embodiments ofthe reagent cartridge may provide or accommodate electroporation devicesthat are not configured as flow-through devices, such as those describedin U.S. Ser. No. 16/147,120 filed 28 Sep. 2018; Ser. No. 16/147,353,filed 28 Sep. 2018; Ser. No. 16/426,310, filed 30 May 2019; Ser. No.16/147,871, filed 30 Sep. 2018; and U.S. Pat. No. 10,323,258, issued 18Jun. 2019, all of which are incorporated by reference in their entiretyfor all purposes.

Another module useful in an automatic multi-module cell processinginstrument is a cell concentration module, which also may be employed toperform buffer exchange to render cells grown in the multi-module cellprocessing instrument electrocompetent. FIG. 3E is a general model 3100of tangential flow filtration. The TFF device operates using tangentialflow filtration, also known as cross-flow filtration. FIG. 1A showscells flowing over a membrane 3104, where the feed flow of the cells3102 in medium or buffer is parallel to the membrane 3104. TFF isdifferent from dead-end filtration where both the feed flow and thepressure drop are perpendicular to a membrane or filter.

FIG. 3F depicts a configuration of a retentate member 3022 (at left), amembrane or filter 3024 (middle), and a permeate member 3020 (at right).In FIG. 3F, retentate member 3022 comprises a tangential flow channel3002, which has a serpentine configuration that initiates at one lowercorner of retentate member 3022—specifically at retentate port3028—traverses across and up then down and across retentate member 3022,ending in the other lower corner of retentate member 3022 at a secondretentate port 3028. Also seen on retentate member 3022 are energydirectors 3091, which circumscribe the region where membrane or filter3024 is seated, as well as interdigitate between areas of the channel.Energy directors 3091 in this embodiment mate with and serve tofacilitate ultrasonic welding or bonding of retentate member 3022 withpermeate/filtrate member 3020 via the energy director component 3091 onpermeate/filtrate member 3020 (at right). Additionally, pin slotalignment elements 3092 are depicted.

Membrane or filter 3024 is seen at center in FIG. 3F, where member 3024is configured to seat within the circumference of energy directors 3091between the retentate member 3022 and the permeate/filtrate member 3020.Permeate/filtrate member 3020 comprises, in addition to energy director3091, through-holes for retentate ports 3028 at each bottom corner(which mate with the through-holes for retentate ports 3028 at thebottom corners of retentate member 3022), as well as a tangential flowchannel 3002 and two permeate/filtrate ports 3026 positioned at the topand center of permeate/filtrate member 3020. The tangential flow channel3002 structure in this embodiment has a serpentine configuration and anundulating geometry, although other geometries may be used. As describedabove, the length of the tangential flow channel is from 10 mm to 1000mm, from 60 mm to 200 mm, or from 80 mm to 100 mm. In some aspects thewidth of the channel structure is from 10 mm to 120 mm, from 40 mm to 70mm, or from 50 mm to 60 mm. In some aspects the cross section of thetangential flow channel 1202 is rectangular, and in some aspects thecross section of the tangential flow channel 1202 is 5 μm to 1000 μmwide and 5 μm to 1000 μm high, 300 μm to 700 μm wide and 300 μm to 700μm high, or 400 μm to 600 μm wide and 400 μm to 600 μm high. In otheraspects, the cross section of the tangential flow channel 1202 iscircular, elliptical, trapezoidal, or oblong, and is 100 μm to 1000 μmin hydraulic radius, 300 μm to 700 μm in hydraulic radius, or 400 μm to600 μm in hydraulic radius.

FIG. 3G is a side perspective view of a reservoir assembly 3050.Reservoir assembly 3050 comprises retentate reservoirs 3052 on eitherside of a single permeate reservoir 3054. Retentate reservoirs 3052 areused to contain the cells and medium as the cells are transferredthrough the cell concentration/growth device or module and into theretentate reservoirs during cell concentration and/or growth.Permeate/filtrate reservoir 3054 is used to collect the filtrate fluidsremoved from the cell culture during cell concentration, or old bufferor medium during cell growth. In this embodiment, there is not a bufferreservoir; instead, buffer or medium is supplied to the retentate memberfrom a reagent reservoir separate from the device module. Additionallyseen in FIG. 3G are grooves 3032 to accommodate pneumatic ports (notseen), a single permeate/filtrate port 3026, and retentate portthrough-holes 3028. The retentate reservoirs are fluidically coupled tothe retentate ports 3028, which in turn are fluidically coupled to theportion of the tangential flow channel disposed in the retentate member(not shown). The permeate/filtrate reservoir is fluidically coupled tothe permeate/filtrate port 3026 which in turn are fluidically coupled tothe portion of the tangential flow channel disposed in permeate/filtratemember (not shown), where the portions of the tangential flow channelsare bifurcated by membrane (not shown). In embodiments including thepresent embodiment, up to 120 mL of cell culture can be grown and/orfiltered, or up to 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30mL or 20 mL of cell culture can be grown and/or concentrated.

FIG. 3H is a side view (left) and a side perspective view (right) of areservoir assembly 3050, which is similar to the reservoir assembly 3050of FIG. 3G. In both views of reservoir assembly 3050 (views in FIGS. 3Gand 3H), the reservoir assembly comprises retentate reservoirs 3052 oneither side of a single permeate reservoir 3054. Retentate reservoirs3052 are used to contain the cells and medium as the cells aretransferred through the cell concentration/growth device or module andinto the retentate reservoirs during cell concentration and/or growth.Permeate/filtrate reservoir 3054 is used to collect the filtrate fluidsremoved from the cell culture during cell concentration, or old bufferor medium during cell growth. In this embodiment, there is not a bufferreservoir; instead in this embodiment, buffer or medium is supplied tothe retentate member from a reagent reservoir separate from the devicemodule. Additionally seen in FIG. 3H are two permeate/filtrate ports3026, and retentate port through-holes 3028. The retentate reservoirsare fluidically coupled to the retentate ports 3028, which in turn arefluidically coupled to the portion of the tangential flow channeldisposed in the retentate member (not shown). The permeate/filtratereservoirs are fluidically coupled to the permeate/filtrate ports 3026which in turn are fluidically coupled to the portion of the tangentialflow channel disposed in permeate/filtrate member (not shown), where theportions of the tangential flow channels are bifurcated by membrane 3024(not shown). In embodiments including the present embodiment, up to 120mL of cell culture can be grown and/or filtered, or up to 100 mL, 90 mL,80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL of cell culture can begrown and/or concentrated.

FIG. 3I depicts a top-down view of the reservoir assemblies 3050 shownin FIG. 3G and FIG. 3H, FIG. 3J depicts a cover 3044 for reservoirassembly 3050 shown in FIG. 3G and FIGS. 3H, and 3K depicts a gasket3045 that in operation is disposed on cover 3044 of reservoir assemblies3050 shown in FIG. 3G and FIG. 3H. FIG. 3I is a top-down view ofreservoir assembly 3050, showing two retentate reservoirs 3052, one oneither side of permeate reservoir 3054. Also seen are grooves 3032 thatwill mate with a pneumatic port (not shown), and fluid channels 3034that reside at the bottom of retentate reservoirs 3052, whichfluidically couple the retentate reservoirs 3052 with the retentateports 3028 (not shown), via the through-holes for the retentate ports inpermeate/filtrate member 3020 and membrane 3024 (also not shown). FIG.3J depicts a cover 3044 that is configured to be disposed upon the topof reservoir assembly 3050. Cover 3044 has round cut-outs at the top ofretentate reservoirs 3052 and permeate/filtrate reservoir 3054. Again,at the bottom of retentate reservoirs 3052 fluid channels 3034 can beseen, where fluid channels 3034 fluidically couple retentate reservoirs3052 with the retentate ports 3028 (not shown). Also shown are threepneumatic ports 3030 for each retentate reservoir 1252 andpermeate/filtrate reservoir 3054. FIG. 3K depicts a gasket 3045 that isconfigured to be disposed upon the cover 3044 of reservoir assembly3050. Seen are three fluid transfer ports 3042 for each retentatereservoir 3052 and for permeate/filtrate reservoir 3054. Again, threepneumatic ports 3030, for each retentate reservoir 3052 and forpermeate/filtrate reservoir 3054, are shown.

FIG. 3L depicts, on the left, an assembled view of the TFF module 3050without retentate member 3022, and on the right, an assembled view ofthe TFF module 3050 with retentate member 3022. Seen are componentsreservoir assembly 3050, a gasket 3045 to be disposed on reservoirassembly 3050, retentate member 3022, membrane or filter 3024, and, onlyseen as a layer beneath membrane 3024, permeate/filtrate member 3020.Also seen are permeate/filtrate ports 3026 (seen at right), which matewith permeate/filtrate ports 3026 on permeate/filtrate reservoir 3054(not seen), as well as two retentate ports 3028, which mate withretentate ports 3028 on retentate reservoirs 3052 (where only oneretentate reservoir 3052 can be seen clearly in this FIG. 3L). Also seenare through-holes for retentate ports 3028 in permeate/filtrate member3020. The left the assembled TFF module 3050 in FIG. 3L typically isfrom 50 to 175 mm in height, or from 75 to 150 mm in height, or from 90to 120 mm in height; from 50 to 175 mm in length, or from 75 to 150 mmin length, or from 90 to 120 mm in length; and is from 30 to 90 mm indepth, or from 40 to 75 mm in depth, or from about 50 to 60 mm in depth.An exemplary TFF device is 110 mm in height, 120 mm in length, and 55 mmin depth.

The TFF device or module depicted in FIGS. 3F-3L can constantly measurecell culture growth, and in some aspects, cell culture growth ismeasured via optical density (OD) of the cell culture in one or both ofthe retentate reservoirs and/or in the flow channel of the TFF device.Optical density may be measured continuously (real-time monitoring) orat specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or so on minutes.Further, the TFF module can adjust growth parameters (temperature,aeration) to have the cells at a desired optical density at a desiredtime. For additional information on TFF modules, please see U.S. Ser.Nos. 62/728,365, filed 7 Sep. 2018; 62/857,599, filed 5 Jun. 2019; and62/867,415, filed 27 Jun. 2019, all of which are incorporated in theentirety for all purposes.

FIGS. 4A-4D depict one module that is useful for both cell growth andfor performing isolation or substantial isolation of cells via bulk gel,as described in relation to FIGS. 2H and 2I. FIG. 4A shows oneembodiment of a rotating growth vial 400 for use with the cell growthdevice described in relation to FIGS. 4B-4D. The rotating growth vial400 is an optically-transparent container having an open end 404 forreceiving liquid media and cells, a central vial region 406 that definesthe primary container for growing cells, a tapered-to-constricted region418 defining at least one light path 410, a closed end 416, and a driveengagement mechanism 412. The rotating growth vial 400 has a centrallongitudinal axis 420 around which the vial rotates, and the light path410 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 410 is positioned in the lower constricted portion ofthe tapered-to-constricted region 418. Optionally, some embodiments ofthe rotating growth vial 400 have a second light path 408 in the taperedregion of the tapered-to-constricted region 418. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 410 is shorter than the second light path 408 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 408 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 412 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 412 such that the rotating growth vial 400 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 400 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 400 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 400 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 400 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 400 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 404with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing instrument. To introduce cells into thevial, a user need only pipette up a desired volume of cells and use thepipette tip to punch through the foil seal of the vial. Open end 404 mayoptionally include an extended lip 402 to overlap and engage with thecell growth device. In automated instruments, the rotating growth vial400 may be tagged with a barcode or other identifying means that can beread by a scanner or camera (not shown) that is part of the automatedinstrument.

The volume of the rotating growth vial 400 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 400 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 400 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 400. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 400 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 4B is a perspective view of one embodiment of a cell growth device430. FIG. 4C depicts a cut-away view of the cell growth device 430 fromFIG. 4B. In both FIGs., the rotating growth vial 400 is seen positionedinside a main housing 436 with the extended lip 402 of the rotatinggrowth vial 400 extending above the main housing 436. Additionally, endhousings 452, a lower housing 432 and flanges 434 are indicated in bothfigures. Flanges 434 are used to attach the cell growth device 430 toheating/cooling means or other structure (not shown). FIG. 4C depictsadditional detail. In FIG. 4C, upper bearing 442 and lower bearing 440are shown positioned within main housing 436. Upper bearing 442 andlower bearing 440 support the vertical load of rotating growth vial 400.Lower housing 432 contains the drive motor 438. The cell growth device430 of FIG. 4C comprises two light paths: a primary light path 444, anda secondary light path 450. Light path 444 corresponds to light path 410positioned in the constricted portion of the tapered-to-constrictedportion of the rotating growth vial 400, and light path 450 correspondsto light path 408 in the tapered portion of the tapered-to-constrictedportion of the rotating growth via 416. Light paths 410 and 408 are notshown in FIG. 4C but may be seen in FIG. 4A. In addition to light paths444 and 440, there is an emission board 448 to illuminate the lightpath(s), and detector board 446 to detect the light after the lighttravels through the cell culture liquid in the rotating growth vial 400.

The motor 438 engages with drive mechanism 412 and is used to rotate therotating growth vial 400. In some embodiments, motor 438 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 438 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 436, end housings 452 and lower housing 432 of the cellgrowth device 430 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 600 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 430 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing instrument.

The processor (not shown) of the cell growth device 430 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 630—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 430, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 4D illustrates a cell growth device 430 as part of an assemblycomprising the cell growth device 430 of FIG. 4B coupled to light source490, detector 492, and thermal components 494. The rotating growth vial400 is inserted into the cell growth device. Components of the lightsource 490 and detector 492 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 432 that houses the motor that rotatesthe rotating growth vial 400 is illustrated, as is one of the flanges634 that secures the cell growth device 430 to the assembly. Also, thethermal components 694 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 430 to the thermal components 494 via the flange 434 on the baseof the lower housing 432. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 400 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 400 by piercing though the foil seal orfilm. The programmed software of the cell growth device 430 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 400. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 400 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring moduletakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 430 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 430 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 430 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional information regarding cell growth modules incorporating arotating growth device, see U.S. Ser. No. 16/360,404, filed 21 Mar. 2019and Ser. No. 16/360,423, filed 21 Mar. 2019, both of which areincorporated by reference in their entirety for all purposes.

When the rotating growth vial RGV) is used as an isolation module, cellsin medium containing 0.25%-6% alginate are transferred into the rotatinggrowth vial by, e.g., a liquid handling system, where first, the cellsare at an appropriate dilution to allow each cell to be isolated orsubstantially isolated from other cells when the medium is gelled, andsecond, the cell colonies that grow from the isolated cells in thegelled or solidified medium are isolated from other cell colonies. Oncethe cells at the proper dilution are loaded into the RGV, solidificationor gelling of the medium is triggered by slowing adding an appropriateamount of, e.g., CaCl₂ dropwise to the RGV, preferably while the RGV isspinning at a low speed. Once the medium is solidifies, the cells can begrown to colonies of terminal size (e.g., normalized) or the cells canbe grown for, e.g., 2-50 doublings, editing is then induced by, e.g.,raising the temperature of the RGV to 42° C. for a period of time toinduce a pL promoter driving transcription of the gRNA, then thetemperature is lowered and the cells are allowed to grow to terminalsize. After the cells have grown to terminal size (e.g., the cells arein senescence and the cell colonies are in no longer increasing insize), the gelled or solidified medium is liquefied by adding anappropriate amount of, e.g., sodium citrate to the solidified mediumdropwise preferably while the RGV is spinning at a low speed. The cellsand medium may then be removed from the RGV by the liquid handlingsystem and filtered in, e.g., a filtration module such as the TFF deviceas described in relation to FIGS. 3F-3L.

FIGS. 5A-5J depict various aspects of a solid wall module configured toperform isolation or substantial isolation of cells in an automatedmulti-module cell processing instrument. FIG. 5A depicts an embodimentof a SWIIN module 550 from an exploded top perspective view. The SWIINmodule 550 in FIG. 5A comprises from the top down, a reservoir gasket orcover 558, a retentate member 504 (where a retentate flow channel cannotbe seen in this FIG. 5A), a perforated member 501 swaged with a filter(filter not seen in FIG. 5A), a permeate member 508 comprisingintegrated reservoirs (permeate reservoirs 552 and retentate reservoirs554), and two reservoir seals 562, which seal the bottom of permeatereservoirs 552 and retentate reservoirs 554. A permeate channel 560 acan be seen disposed on the top of permeate member 508, defined by araised portion 576 of serpentine channel 560 a, and ultrasonic tabs 564can be seen disposed on the top of permeate member 508 as well. Theperforations that form the wells on perforated member 501 are not seenin this FIG. 5A; however, through-holes 566 to accommodate theultrasonic tabs 564 are seen. In addition, supports 570 are disposed ateither end of SWIIN module 550 to support SWIIN module 550 and toelevate permeate member 508 and retentate member 504 above reservoirs552 and 554 and to minimize bubbles or air entering the fluid path fromthe permeate reservoir to serpentine channel 560 a or the fluid pathfrom the retentate reservoir to serpentine channel 560 b (neither fluidpath is seen in this FIG. 5A, but see FIG. 5H).

In this FIG. 5A, it can be seen that the serpentine channel 560 a thatis disposed on the top of permeate member 508 traverses permeate member508 for most of the length of permeate member 508 except for the portionof permeate member 508 that comprises permeate reservoirs 552 andretentate reservoirs 554 and for most of the width of permeate member508. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types, the pore sizes can be ashigh as 0.5 μm. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

FIG. 5B is a top-down view of permeate member 508, showing serpentinechannel 560 a (the portion of the serpentine channel disposed inpermeate member 508) defined by raised portion 576 of serpentine channel560 a, permeate reservoirs 552, retentate reservoirs 554, reservoirports 556 (two of the four of which are labeled), ultrasonic tabs 564disposed at each end of permeate member 508 and on the raised portion576 of serpentine channel 560 a of permeate member 408, and two permeateports 511 and two retentate ports 507 are also seen.

FIG. 5C is a bottom-up view of retentate member 504, showing serpentinechannel 560 b (the portion of the serpentine channel disposed inretentate member 508) defined by the raised portion 576 of theserpentine channel 560 b. Also seen is an integrated reservoir cover 578for the permeate and retentate reservoirs that mate with permeatereservoirs 552 and retentate reservoirs 554 on the permeate member. Theintegrated reservoir cover 578 comprises reservoir access apertures 532a, 532 b, 532 c, and 532 d, as well as pneumatic ports 533 a, 533 b, 533c and 533 d. As with previous embodiments, the serpentine channel 560 aof permeate member 508 and the serpentine channel 560 b of retentatemember 504 mate to form the top (retentate member) and bottom (permeatemember) of a mated serpentine channel. The footprint length of theserpentine channel structure is from, e.g., from 80 mm to 500 mm, from100 mm to 400 mm, or from 150 mm to 250 mm. In some aspects, the entirefootprint width of the channel structure is from 50 mm to 200 mm, from75 mm to 175 mm, or from 100 mm to 150 mm.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 10 mm to 12mm in hydraulic radius.

As in previous embodiments, disposed between serpentine channels 560 aand 560 b is perforated member 501 (adjacent retentate member 504) andfilter 503 (adjacent permeate member 508), where filter 503 is swagedwith perforated member 501. Serpentine channels 560 a and 560 b can haveapproximately the same volume or a different volume. For example, each“side” or portion 560 a, 560 b of the serpentine channel may have avolume of, e.g., 2 mL, or serpentine channel 560 a of permeate member508 may have a volume of 2 mL, and the serpentine channel 560 b ofretentate member 504 may have a volume of, e.g., 3 mL. The volume offluid in the serpentine channel may range from about 2 mL to about 80mL, or about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL(note these volumes apply to a SWIIN module comprising a, e.g., 50-500Kperforation member).

The serpentine channel portions 560 a and 560 b of the permeate member508 and retentate member 504, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness. As inpreviously described embodiments the retentate (and permeate) membersmay be fabricated from PMMA (poly(methyl methacrylate) or othermaterials may be used, including polycarbonate, cyclic olefin co-polymer(COC), glass, polyvinyl chloride, polyethylene, polyamide,polypropylene, polysulfone, polyurethane, and co-polymers of these andother polymers. Preferably at least the retentate member is fabricatedfrom a transparent material so that the cells can be visualized (see,e.g., FIG. 5I and the description thereof). For example, a video camera(as described supra in relation to FIG. 2G and infra in relation to FIG.5I) may be used to monitor cell growth by, e.g., density changemeasurements based on an image of an empty well, with phase contrast, orif, e.g., a chromogenic marker, such as a chromogenic protein, is usedto add a distinguishable color to the cells. Chromogenic markers such asblitzen blue, dreidel teal, virginia violet, vixen purple, prancerpurple, tinsel purple, maccabee purple, donner magenta, cupid pink,seraphina pink, scrooge orange, and leor orange (the Chromogenic ProteinPaintbox, all available from ATUM (Newark, Calif.)) obviate the need touse fluorescence, although fluorescent cell markers, fluorescentproteins, and chemiluminescent cell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 550 may be controlled by, e.g., moving heated air over the top of(e.g., over the top surface of retentate member) of the SWIIN module550, or by applying a transparent heated lid over at least theserpentine channel portion 560 b of the retentate member 504. See, e.g.,FIG. 5I and the description thereof infra.

As with the embodiments described previously, in SWIIN module 550 cellsand medium—at a dilution appropriate for Poisson or substantial Poissondistribution of the cells in the microwells of the perforated member—areflowed into serpentine channel 560 b from ports in retentate member 504,and the cells settle in the microwells while the medium passes throughthe filter into serpentine channel 560 a in permeate member 508. Thecells are retained in the microwells of perforated member 501 as thecells cannot travel through filter 503. Appropriate medium may beintroduced into permeate member 508 through permeate ports 511. Themedium flows upward through filter 503 to nourish the cells in themicrowells (perforations) of perforated member 501. Additionally, bufferexchange can be effected by cycling medium through the retentate andpermeate members. In operation, the cells are deposited into themicrowells, are grown for an initial, e.g., 2-100 doublings, editing isinduced by, e.g., raising the temperature of the SWIIN to 42° C. toinduce a temperature inducible promoter or by removing growth mediumfrom the permeate member and replacing the growth medium with a mediumcomprising a chemical component that induces an inducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module550 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 560 a and thus to filter 503 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 5D is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 5D, it canbe seen that serpentine channel 560 a is disposed on the top of permeatemember 508 is defined by raised portions 576 and traverses permeatemember 508 for most of the length and width of permeate member 508except for the portion of permeate member 508 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 552can be seen). Moving from left to right, reservoir gasket 558 isdisposed upon the integrated reservoir cover 578 (cover not seen in thisFIG. 5D) of retentate member 504. Gasket 558 comprises reservoir accessapertures 532 a, 532 b, 532 c, and 532 d, as well as pneumatic ports 533a, 533 b, 533 c and 533 d. Also at the far left end is support 570.Disposed under permeate reservoir 552 can be seen one of two reservoirseals 562. In addition to the retentate member being in cross section,the perforated member 501 and filter 503 (filter 503 is not seen in thisFIG. 5D) are in cross section. Note that there are a number ofultrasonic tabs 564 disposed at the right end of SWIIN module 550 and onraised portion 576 which defines the channel turns of serpentine channel560 a, including ultrasonic tabs 564 extending through through-holes 566of perforated member 501. There is also a support 570 at the end distalreservoirs 552, 554 of permeate member 508.

FIG. 5E is a side perspective view of an assembled SWIIIN module 550,including, from right to left, reservoir gasket 558 disposed uponintegrated reservoir cover 578 (not seen) of retentate member 504.Gasket 558 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 558 comprises reservoir access apertures 532 a, 532 b, 532 c, and532 d, as well as pneumatic ports 533 a, 533 b, 533 c and 533 d. Also atthe far left end is support 570 of permeate member 508. In addition,permeate reservoir 552 can be seen, as well as one reservoir seal 562.At the far right end is a second support 570.

FIG. 5F is a side perspective view of the reservoir portion of permeatemember 508 and retentate member 504, including gasket 558. Seen arepermeate reservoirs 552 as the outside reservoirs, and retentatereservoirs 554 between permeate reservoirs 552. It should be apparent toone of ordinary skill in the art given the present description, however,that this particular configuration of reservoirs may be changed withpermeate 552 and retentate 554 reservoirs alternating in position; withboth permeate reservoirs 552 on one side of SWIIN module 550 and bothretentate reservoirs 554 on the other side of SWIIN module 550, or theretentate reservoirs 554 may be positioned at the two sides with thepermeate reservoirs 552 between the retentate reservoirs. Again, gasket558 comprises reservoir access apertures 532 a, 532 b, 532 c, and 532 d,as well as pneumatic ports 533 a, 533 b, 533 c and 533 d. In addition,two reservoir seals 562 can be seen, each sealing one permeate reservoir552 and one retentate reservoir 554. Also seen is support 570 at the“reservoir end” of permeate member 508.

FIG. 5G is a side perspective cross sectional view of permeate reservoir552 of permeate member 508 and retentate member 504 and gasket 558.Reservoir access aperture 532 c and pneumatic aperture 533 c can beseen, as well as support 570. Also seen is perforated member 501 andfilter 503 (filter 503 is not seen clearly in this FIG. 5G but issandwiched in between perforated member 501 and permeate member 508). Afluid path 572 from permeate reservoir 552 to serpentine channel 560 ain permeate member 508 can be seen, as can reservoir seal 562.

FIG. 5H is a small segment of a cross section of SWIIN module 550,showing the retentate member 504, perforated member 501, filter 503, andretentate member 508. FIG. 5H also shows a fluid path 572 from apermeate reservoir to the serpentine channel 560 a disposed in permeatemember 508, and a fluid path 574 from a retentate reservoir to theserpentine channel 560 b disposed in permeate member 504. As mentionedpreviously, the reservoir architecture of this embodiment isparticularly advantageous as bubbling is minimized. That is, because thereservoirs and reservoir ports are positioned below the retentate andpermeate serpentine channels, there is no instantaneous flow of fluid inthe reservoirs into channels that connect the reservoir ports to theretentate and permeate channels. Instead, flow is controlled by theapplication of pressure (positive or negative) and an appropriate timechosen by the user.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (SWIIN top) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, recovercells from specific wells (e.g., slow-growing cell colonies), or use,e.g., UV light to irradiate specific wells (e.g., fast-growing cellscolonies). Imaging may also be used to assure proper fluid flow in theserpentine channel 560.

FIG. 5I depicts the embodiment of the SWIIN module in FIGS. 5A-5Hfurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 598 comprises a SWIIN module 550seen lengthwise in cross section, where one permeate reservoir 552 isseen. Disposed immediately upon SWIIN module 550 is cover 594 anddisposed immediately below SWIIN module 550 is backlight 580, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 582, which is disposed over a heatsink 584. In thisFIG. 5I, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 586 and heat sink 588, as well as twothermoelectric coolers 592, and a controller 590 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability.

FIG. 5J is an exemplary pneumatic block diagram suitable for the SWIINmodule depicted in FIGS. 5A-5I. In this configuration, there are twomanifold arms that are controlled independently and there are twoproportional valves, one each for the manifold arms. Tables 1-3 relateto the pneumatic diagram in FIG. 5J. Table 1 lists, for each step 1-32,the manifold arm status (open=arm open, closed=arm closed, motor engagedfor pressurization); pump status (1: on, 0: off); energy status (1:energized, 0: de-energized) for each solenoid valve 1-4; and thepressure in psi for each proportional valve. Table 2 lists, for eachstep 1-32, the detection and threshold status for flow meters 1 and 2 aswell as the duration of each step. When a change in pressure precedes avalve event, there is a delay of 1 second after reaching the set pointbefore energizing the valves to avoid applying over- and under-shoots tothe system. FALL=monitor for a falling signal, RISE=monitor for a risingsignal. “Requires pLLD”=requires pressure-driven liquid level detection,such as, e.g., via air-displacement pipettor. Table 3 lists, for eachstep 1-32, the volumes for each reservoir, permeate reservoirs 1 and 2,and retentate reservoirs 1 and 2; the temperature of the SWIIN; andnotes for operation.

FIG. 6A is a block diagram of one embodiment of a method 400 for usingthe automated multi-module cell processing instrument of FIG. 3A,including the enrichment modules configured to perform the workflowsdescribed in relation to FIGS. 2A-2K. In a first step, cells aretransferred 601 from reagent cartridge 310 (please refer to FIG. 3Aregarding element numbers 300) to growth vial 318. The cells areincubated 602, e.g., until they grow to a desired OD 603. The cells arethen transferred 604 to cell concentration module 322 to perform mediumor buffer exchange and render the cells competent (e.g.,electrocompetent) via medium/buffer exchange while also reducing thevolume of the cell sample to a volume appropriate for electroporation,as well as to remove unwanted components, e.g., salts, from the cellsample. Once the cells have been rendered competent and suspended in anappropriate volume for transformation, the cell sample is transferred612 to flow-through electroporation device 330 (transformation module)in reagent cartridge 310.

While cells are being processed for electroporation, automatedmulti-module cell processing instrument 300 may store the nucleic acidsto be electroporated into the cells 611 where the editing vector is thentransferred to the transformation module 612. The editing vector and thecells are thus combined in flow-through electroporation device 330 andthe flow-through electroporation device is engaged 613.

After electroporation, the transformed cells optionally are transferred614 to an isolation device 614 to, e.g., to be isolated, recover fromthe transformation process, be subjected to selection, and for, in thisparticular example, genome editing and colony normalization. Once thetransformed cells have been enriched or selected, the enriched/selectedcells may then be subjected to further editing 615, where all or some ofsteps 601-614 may be repeated, or the cells may be used in research 616.

FIG. 6B is a block diagram of one embodiment of a method 606 for usingthe automated multi-module cell processing instrument of FIG. 3A,including the solid wall isolation/growth/editing/normalization modulesconfigured to perform the workflow described in relation to FIGS. 2J and2K. In a first step, cells are transferred 601 from reagent cartridge310 (refer to FIG. 3A) to growth vial 318. The cells are incubated 602,e.g., until they grow to a desired OD 603. The cells are thentransferred 604 to cell concentration module 322 (such as a cellconcentration module comprising a filter) to perform medium or bufferexchange and render the cells competent (e.g., electrocompetent) viamedium/buffer exchange while also reducing the volume of the cell sampleto a volume appropriate for electroporation, as well as to removeunwanted components, e.g., salts, from the cell sample. Once the cellshave been rendered competent and suspended in an appropriate volume fortransformation, the cell sample is transferred 612 to flow-throughelectroporation device 330 (transformation module) in reagent cartridge310.

While cells are being processed for electroporation, automatedmulti-module cell processing instrument 300 may store the nucleic acidsto be electroporated into the cells 611 where the editing vector is thentransferred to the transformation module 612. The editing vector and thecells are thus combined in flow-through electroporation device 330 andthe flow-through electroporation device is engaged 613.

After electroporation, the transformed cells are transferred 617 to anisolation device to, e.g., recover from the transformation process, besubjected to selection, be isolated, grown, induced, and edited.Alternatively, after electroporation, the transformed cells may betransferred to a recovery module, where the cells are allowed torecover, selection may take place, and the cells can be diluted, ifnecessary, so that when introduced into the isolation device, the cellsisolated according to a Poisson distribution or a substantial Poissondistribution. Once the transformed cells have recovered, been selected(e.g., by an antibiotic or other reagent added from the reagentcartridge), been isolated and grown a desired number of doublings. Atthis point, editing has been induced and the size of the colonies aremonitored 618 and slow-growing colonies are selected. The cells now maybe used in research 620 or the cells may then be subjected to furtherediting 619, where all or some of steps 601-618 may be repeated.

FIG. 6C is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall isolation/growth/editing/normalization module for enrichment foredited cells. The cell processing instrument 630 may include a housing674, a reservoir of cells to be transformed or transfected 632, and agrowth module (a cell growth device) 634. The cells to be transformedare transferred from a reservoir to the growth module to be cultureduntil the cells hit a target OD. Once the cells hit the target OD, thegrowth module may cool or freeze the cells for later processing, or thecells may be transferred to a filtration module 660 where the cells arerendered electrocompetent and concentrated to a volume optimal for celltransformation. Once concentrated, the cells are then transferred to theelectroporation device 638 (e.g., transformation/transfection module).Exemplary electroporation devices of use in the automated multi-modulecell processing instruments for use in the multi-module cell processinginstrument include flow-through electroporation devices such as thosedescribed in U.S. Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No.16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018;and Ser. No. 16/147,871, filed 30 Sep. 2018 all of which are hereinincorporated by reference in their entirety.

In addition to the reservoir for storing the cells, the system 630 mayinclude a reservoir for storing editing oligonucleotide cassettes 646and a reservoir for storing an expression vector backbone 648. Both theediting oligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module650, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 652 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 646 or 648. Once the processes carried out by thepurification module 652 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 638, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via filtration module 660. In electroporation device638, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 640.

Following recovery, and, optionally, selection, the cells aretransferred to an isolation, growth, induction, editing, andnormalization module 670, where the cells are diluted andcompartmentalized such that there is an average of one cell percompartment. Isolation can entail a well (See FIGS. 2J and 2K), adroplet (see FIGS. 2F and 2G), in gel in three-dimensional space (seeFIGS. 2H and 2I), or an area, e.g., cells isolated from one another onan agar plate or arrayed on a functionalized substrate (FIGS. 2A-2E).Once isolated, the cells are allowed to grow for a pre-determined numberof doublings. Once these initial colonies are established, editing isinduced and the edited cells are allowed to establish colonies, whichare grown to terminal size (e.g., the colonies are normalized). In someembodiments, editing is induced by one or more of the editing componentsbeing under the control of an inducible promoter. In some embodiments,the inducible promoter is activated by a rise in temperature and“deactivated” by lowering the temperature.

The recovery, selection, isolation, induction, editing and growthmodules may all be separate, may be arranged and combined as shown inFIG. 3A, or may be arranged or combined in other configurations. Incertain embodiments, recovery and selection are performed in one module,and isolation, growth, editing, and normalization are performed in aseparate module. Alternatively, recovery, selection, isolation, growth,editing, and normalization are performed in a single module.

Once the normalized cell colonies are pooled, the cells may be stored,e.g., in a storage module 642, where the cells can be kept at, e.g., 4°C. until the cells are retrieved for further study. Alternatively, thecells may be used in another round of editing. The multi-module cellprocessing instrument is controlled by a processor 672 configured tooperate the instrument based on user input, as directed by one or morescripts, or as a combination of user input or a script. The processor672 may control the timing, duration, temperature, and operations of thevarious modules of the system 630 and the dispensing of reagents. Forexample, the processor 672 may cool the cells post-transformation untilediting is desired, upon which time the temperature may be raised to atemperature conducive of genome editing and cell growth. The processormay be programmed with standard protocol parameters from which a usermay select, a user may specify one or more parameters manually or one ormore scripts associated with the reagent cartridge may specify one ormore operations and/or reaction parameters. In addition, the processormay notify the user (e.g., via an application to a smart phone or otherdevice) that the cells have reached the target OD as well as update theuser as to the progress of the cells in the various modules in themulti-module instrument.

The automated multi-module cell processing instrument 630 is anuclease-directed genome editing instrument and can be used in singleediting systems (e.g., introducing one or more edits to a cellulargenome in a single editing process). The system of FIG. 6D, describedbelow, is configured to perform sequential editing, e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell; and/or recursive editing, e.g. utilizing asingle nuclease-directed system to introduce sequentially two or moregenome edits in a cell.

FIG. 6D illustrates another embodiment of a multi-module cell processinginstrument. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. As with the embodimentshown in FIG. 6C, the cell processing instrument 680 may include ahousing 674, a reservoir for storing cells to be transformed ortransfected 632, and a cell growth module (comprising, e.g., a rotatinggrowth vial) 634. The cells to be transformed are transferred from areservoir to the cell growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing or transfer the cells to afiltration module 660 where the cells are subjected to buffer exchangeand rendered electrocompetent, and the volume of the cells may bereduced substantially. Once the cells have been concentrated to anappropriate volume, the cells are transferred to electroporation device638. In addition to the reservoir for storing cells, the multi-modulecell processing instrument includes a reservoir for storing an editingvector pre-assembled with editing oligonucleotide cassettes 682. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 638, which already contains the cell culturegrown to a target OD. In the electroporation device 638, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 686, where thecells are allowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 642,where the cells can be stored at, e.g., 4° C. for later processing, orthe cells may be diluted and transferred to a selection/isolation(singulation)/growth/editing/normalization module 688. In theisolation/edit/growth module 688, the cells are arrayed such that thereis an average of one cell per microwell. The arrayed cells may be inselection medium to select for cells that have been transformed ortransfected with the editing vector(s). Once isolated, the cells areallowed to grow through 2-200 doublings and establish colonies. Oncecolonies are established, editing is induced by providing conditions(e.g., temperature, addition of an inducing or repressing chemical) toinduce editing. Once editing is initiated and allowed to proceed, thecells are allowed to grow to terminal size (e.g., normalization of thecolonies) and then can be flushed out of the microwells and pooled, thentransferred to the storage (or recovery) unit 642 or can be transferredto a growth module 634 for another round of editing. In between poolingand transfer to a growth module, there may be one or more additionalsteps, such as cell recovery, medium exchange, cells concentration,etc., by, e.g., filtration. Note that theselection/isolation/growth/induction/editing and normalization modulesmay be the same module, where all processes are performed in the module,or selection and/or dilution may take place in a separate vessel beforethe cells are transferred to the isolation/growth/editing/normalizationmodule. Once the putatively-edited cells are pooled, they may besubjected to another round of editing, beginning with growth, cellconcentration and treatment to render electrocompetent, andtransformation by yet another donor nucleic acid in another editingcassette via the electroporation module 638.

In electroporation device 638, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument exemplified in FIG. 6D iscontrolled by a processor 672 configured to operate the instrument basedon user input or is controlled by one or more scripts including at leastone script associated with the reagent cartridge. The processor 672 maycontrol the timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe system 680. For example, a script or the processor may control thedispensing of cells, reagents, vectors, and editing oligonucleotides;which editing oligonucleotides are used for cell editing and in whatorder; the time, temperature and other conditions used in the recoveryand expression module, the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor may be programmed to notify a user (e.g., via anapplication) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 6D, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.

In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid.

FIG. 6E illustrates another embodiment of a multi-module cell processingsystem. This embodiment depicts an exemplary system that 1) includesediting induction and cell selection in addition to enrichment, and 2)performs recursive gene editing on a cell population. As with theembodiments shown in FIGS. 6C and 6D, the cell processing system 690 mayinclude a housing 674, a reservoir for storing cells to be transformedor transfected 632, and a cell growth module (a cell growth device) 634.The cells to be transformed are transferred from a reservoir to the cellgrowth module to be cultured until the cells hit a target OD. Once thecells hit the target OD, the growth module may cool or freeze the cellsfor later processing or transfer the cells to an optional filtrationmodule 660 where the cells are rendered electrocompetent, and the volumeof the cells may be reduced substantially. Once the cells have beenconcentrated to an appropriate volume, the cells are transferred toelectroporation device 638. In addition to the reservoir for storingcells, the multi-module cell processing system includes a reservoir forstoring the vector comprising editing oligonucleotides 682. Theassembled nucleic acids are transferred to the electroporation device638, which already contains the cell culture grown to a target OD. Inthe electroporation device 638, the nucleic acids are electroporatedinto the cells. Following electroporation, the cells are transferredinto an optional recovery module 686, where the cells are allowed torecover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 642,where the cells can be stored at, e.g., 4° C. until the cells areretrieved for further study, or the cells may be transferred to anisolation and growth module 634. In the isolation (e.g., singulation)and growth module 689, the cells are arrayed such that there is anaverage of one cell per compartment. In some embodiments, a compartmentmay be a well (see FIGS. 2J and 2K); a droplet (see FIGS. 2F and 2G); ingel in three-dimensional space (see FIGS. 2H and 2I); or an area, e.g.,cells isolated from one another on an agar plate or arrayed on, e.g., afunctionalized substrate (see FIGS. 2A, 2B, 2D, and 2E). Once isolated,the cells are allowed to grow through 2-50 doublings or more andestablish colonies. Once colonies are established, the substrate withthe cell colonies is transferred to an induction module, whereconditions exist (temperature, addition of an inducing or repressingchemical) to induce editing. Once editing is initiated and allowed toproceed, the substrate is transferred to a cherry picking module 692,which may include, e.g., a colony measuring and picking device thatselects small colonies of cells; a spectrophotometer or video cameraconfigured to measure OD in wells or droplets and collect colonies ofedited cells or ablate or irradiate colonies of unedited cells based oncell growth; or a spectrophotometer configured to measure other cellularcharacteristics in wells or droplets and collect colonies of editedcells based on cell characteristics that correlate with cell growth.Note that the isolation and growth module and cherry-picking module maybe linked. Once the putatively-edited cells are selected (or putativelyun-edited cells are eliminated), the edited cells may be subjected toanother round of editing, beginning with transformation by yet anotherdonor nucleic acid in another editing cassette via the electroporationmodule 638.

In electroporation device 638, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., donor nucleicacids. The multi-module cell processing system exemplified in FIG. 6E iscontrolled by a processor 672 configured to operate the instrument basedon user input or is controlled by one or more scripts including at leastone script associated with the reagent cartridge. The processor 672 maycontrol the timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe system 690. For example, a script or the processor may control thedispensing of cells, reagents, vectors, and editing oligonucleotides;which editing oligonucleotides are used for cell editing and in whatorder; the time, temperature and other conditions used in the recoveryand expression module, the wavelength at which OD is read in the cellgrowth module, the target OD to which the cells are grown, and thetarget time at which the cells will reach the target OD. In addition,the processor may be programmed to notify a user (e.g., via anapplication) as to the progress of the cells in the automatedmulti-module cell processing system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Other equivalent methods, steps and compositionsare intended to be included in the scope of the invention. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Celsius, and pressure is at or near atmospheric.

Example 1: Enrichment of Editing Cells by Growth Lag Identification

Transformation:

100 ng of the editing vector cloned library or Gibson Assembly® reactionwas transformed by electroporation into 100 μL competent EC1 cellscontaining the engine vector. The electroporator was set to 2400 V in 2mm cuvette. Following transformation, the cells were allowed to recoverfor 3 hours in SOB medium. A 10-fold dilution series of recovered cells(in H₂O) was spot plated and the resulting CFU counts/dilution ratioswere used to calculate transformation efficiency.

Plating and Colony Arraying:

100 μL of the appropriate dilution was plated on LB medium+25 μg/mLchlor and +arabinose and grown at 30° C. for 6-8 hours. Alternatively,the cells may be grown in liquid culture in LB medium+25 μg/mL chlor at30° C. for 6-8 hours. The temperature of the plate was adjusted to 42°C. and the plates were incubated for two hours. The temperature was thenadjusted back to 30° C. and the cells were allowed to recover overnight.

Edited Cell Identification:

Small-size colonies were identified. The small colony-size phenotypeindicates cell viability was compromised during the induced-editingprocedure. Efficient recovery of edited cells from the initial pool wasaccomplished by identifying and picking small colonies and arrayingcells from these small colonies onto a 96-well plate to create a libraryof edited cells, or the cells from the colonies were pooled generatinghighly-edited cell populations for recursive editing. Editing wasassessed/validated by sequencing. It was found that 85% of the smallcolonies were edited cells (data not shown).

Example 2: Assessing Editing by Optical Density

FIG. 2C is a depiction of the growth profiles of randomly-pickedvariants from a silent PAM mutation (SPM) library. This 500-memberlibrary targets regions located across the entire E. coli genome andintegrated synonymous mutations that have no expected fitness effects.Colonies were picked from agar plates of uninduced transformed cells.Cells were picked from an agar plate and grown up in 200 μLLB+chlor/carb overnight in a 96-well microtiter plate format. 10 μL ofthe well content of the parent microtiter plate was then transferred totwo replica daughter microtiter plates that received either no induction(top) or gRNA and nuclease induction (via the pL inducible promoter) for1 hour at 42° C. (bottom). The well maps show the relative OD at 6hours; the full growth curves are shown for reference. The replica wellsrepresent growth observed from the same cassette design with or withoutgRNA induction. While the majority of the wells for the no-inductionplate show normal growth profiles, the induced plate shows that a largefraction of the gRNA designs are still active when induced, indicated bya large lag phase before the cells reach exponential growth. That is,the actively-editing cells have reduced viability due to DNA damage suchthat many cells in the colonies die off, and those edited cells that dosurvive take longer to re-establish colonies. This characteristic ofedited cells can be exploited to screen for active editing in ahigh-throughput manner.

Example 3: Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation:

5 nM of oligonucleotides synthesized on a chip were amplified using Q5polymerase in 50 μL volumes. The PCR conditions were 95° C. for 1minute; 8 rounds of 95° C. for 30 seconds/60° C. for 30 seconds/72° C.for 2.5 minutes; with a final hold at 72° C. for 5 minutes. Followingamplification, the PCR products were subjected to SPRI cleanup, where 30μL SPRI mix was added to the 50 μL PCR reactions and incubated for 2minutes. The tubes were subjected to a magnetic field for 2 minutes, theliquid was removed, and the beads were washed 2× with 80% ethanol,allowing 1 minute between washes. After the final wash, the beads wereallowed to dry for 2 minutes, 50 μL 0.5×TE pH 8.0 was added to thetubes, and the beads were vortexed to mix. The slurry was incubated atroom temperature for 2 minutes, then subjected to the magnetic field for2 minutes. The eluate was removed and the DNA quantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation:

A 10-fold serial dilution series of purified backbone was performed, andeach of the diluted backbone series was amplified under the followingconditions: 95° C. for 1 minute; then 30 rounds of 95° C. for 30seconds/60° C. for 1.5 minutes/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. After amplification, the amplified backbone wassubjected to SPRI cleanup as described above in relation to thecassettes. The backbone was eluted into 100 μL ddH₂O and quantifiedbefore isothermal nucleic acid assembly.

Isothermal Nucleic Acid Assembly:

150 ng backbone DNA was combined with 100 ng cassette DNA. An equalvolume of 2× isothermal nucleic acid assembly master mix was added, andthe reaction was incubated for 45 minutes at 50° C. After assembly, theassembled backbone and cassettes were subjected to SPRI cleanup, asdescribed above.

Transformation of Editing Vector into E Cloni®:

20 μL of the prepared editing vector isothermal assembly reaction wasadded to 30 μL chilled water along with μL E Cloni® (Lucigen, Middleton,Wis.) supreme competent cells. An aliquot of the transformed cells werespot plated to check the transformation efficiency, where >100× coveragewas required to continue. The transformed E Cloni® cells were outgrownin 25 mL SOB+100 μg/mL carbenicillin (carb). Glycerol stocks weregenerated from the saturated culture by adding 500 μL 50% glycerol to1000 μL saturated overnight culture. The stocks were frozen at −80° C.This step is optional, providing a ready stock of the cloned editinglibrary. Alternatively, Gibson or another assembly of the editingcassettes and the vector backbone can be performed before each editingexperiment.

Creation of New Cell Line Transformed with Engine Vector:Transformation:

1 μL of the engine vector DNA (comprising a coding sequence for MAD7nuclease under the control of the pL inducible promoter, achloramphenicol resistance gene, and the λ Red recombineering system)was added to 50 μL EC1 strain E. coli cells. The transformed cells wereplated on LB plates with 25 μg/mL chloramphenicol (chlor) and incubatedovernight to accumulate clonal isolates. The next day, a colony waspicked, grown overnight in LB+25 μg/mL chlor, and glycerol stocks wereprepared from the saturated overnight culture by adding 500 μL 50%glycerol to 1000 μL culture. The stocks of EC1 comprising the enginevector were frozen at −80° C.

Preparation of Competent Cells:

A 1 mL aliquot of a freshly-grown overnight culture of EC1 cellstransformed with the engine vector was added to a 250 mL flaskcontaining 100 mL LB/SOB+25 μg/mL chlor medium. The cells were grown to0.4-0.7 OD, and cell growth was halted by transferring the culture toice for 10 minutes. The cells were pelleted at 8000×g in a JA-18 rotorfor 5 minutes, washed 3× with 50 mL ice cold ddH₂O or 10% glycerol andpelleted at 8000×g in JA-18 rotor for 5 minutes. The washed cells wereresuspended in 5 mL ice cold 10% glycerol and aliquoted into 200 μLportions. Optionally at this point the glycerol stocks could be storedat −80° C. for later use.

Example 4: Exemplary Workflow for Diversity Generation

First, a library was designed, taking approximately 1-4 weeks. Thereagents were manufactured (approximately 2 weeks) and received.Optionally, the engine and editing vectors are pre-assembled, althoughin some workflows, the engine vector is pre-assembled and used totransform the cells to be edited in advance, while the editing vector istransformed into the cells in the automated multi-module cell processinginstrument. Yet another option is to combine the engine and editingvectors into a single vector. In this example, both the nuclease and theguide nucleic acid were under the control of the pL inducible promoter,which is induced by temperature.

Transformation in the automated transformation module employing anelectroporation device took approximately 5 minutes. At this point, thetransformed cells may optionally be stored in a freezer for laterexperiments, or the cells can proceed to editing. The cells wereoutgrown to saturation (approximately 12 hours) and then diluted andplated to agar, such as on 3-4 Q trays. The plated cells were allowed togrow for 6-12 hours, and then are induced by raising the temperature to42° C. In one option, the cells were grown to colonies of terminal size,and then the colonies were harvested and an aliquot of the cells isprepared for sequencing to QC the library. The cells on the Q trays werethen picked into 96-well plates. In one option, all colonies werepicked. In an alternative option, only small colonies were picked. Oncepicked, the colonies are allowed to grow overnight, and aliquots fromthese colonies are replica plated into a different 96-well plate. Atthis point, assays can be run to identify edits.

Alternatively, the cells on the Q trays may be scraped and pooled into atube. A glycerol stock can be made of this library, and another aliquotcan be used to run selections on, e.g., Q trays. The results of theediting process are then analyzed using amplicon verification or genomevalidation.

Example 5: Exemplary Workflow for Combinatorial Edit Generation

As with the diversity workflow, a library was designed, takingapproximately 1-4 weeks. The reagents were manufactured (approximately 2weeks) and received. Optionally, the engine and editing vectors arepre-assembled, although in some workflows, the engine vector ispre-assembled and used to transform the cells to be edited in advance,while the editing vector is transformed into the cells in the automatedmulti-module cell processing instrument. Yet another option is tocombine the engine and editing vectors into a single vector. In thisembodiment, both the nuclease and the guide nucleic acid are under thecontrol of the pL inducible promoter, which is induced by temperature.

Transformation in the automated transformation module employing anelectroporation device took approximately 5 minutes. At this point, thetransformed cells may optionally be stored in a freezer for laterexperiments, or the cells can proceed to editing. The cells can then beoutgrown to saturation (approximately 12 hours) and then diluted andplated to agar, such as on 3-4 QTRAYS™ (Molecular Devices, San Jose,Calif.). The plated cells were allowed to grow for 6-12 hours, and thenwere induced by raising the temperature to 42° C. In one option, thecells are grown to colonies of terminal size. Next, the cells aresubjected to an optional recursion step, in which the plates arescraped, pooled, cured, made electrocompetent, and transformed withanother set of editing vectors.

In one option, the cells were grown to colonies of terminal size, andthen the colonies were harvested and an aliquot of the cells wasprepared for sequencing to QC the library. The cells on the QTRAYS™ arethen picked into 96-well plates. In one option, all colonies are picked.In an alternative option, only small colonies are picked. Once picked,the colonies are allowed to grow overnight and aliquots from thesecolonies are replica plated into a different 96-well plate. At thispoint, assays can be run to identify edits.

Alternatively, the cells on the QTRAYS™ may be scraped and pooled into atube. A glycerol stock can be made of this library, and another aliquotcan be used to run selections on, e.g., QTRAYS™. The results of theediting process are then analyzed using amplicon verification or genomevalidation.

Example 6: Exemplary Workflow for Diversity Generation with CellPrinting

As in the workflows described above, first a library was designed,taking approximately 1-4 weeks. The reagents were manufactured(approximately 2 weeks) and received. Optionally, the engine and editingvectors are pre-assembled, although in some workflows, the engine vectoris pre-assembled and used to transform the cells to be edited inadvance, while the editing vector is transformed into the cells in theautomated multi-module cell processing instrument. Yet another option isto combine the engine and editing vectors. In this embodiment, both thenuclease and the guide nucleic acid were under the control of the pLinducible promoter, which is induced by temperature.

Transformation in the automated transformation module employing anelectroporation device took approximately 5 minutes. At this point, thetransformed cells may optionally be stored in a freezer for laterexperiments, or the cells can proceed to editing. The cells can then beoutgrown to saturation (approximately 12 hours). Once grown, the cellsare printed 100×96-well or 25×384-well plates. The number of platesgrows with lower basal editing efficiency (e.g., 50% editing efficiencyrequires 200×96-well plates). The cells are allowed to grow for severalto many doublings and are then the promoters driving both transcriptionof the nuclease and guide nucleic acid are induced with temperature.After induction, the temperature is lowered and the edited cells areallowed to grow for some time. If wells have high degree ofpolyclonality they optionally can be repooled and run back through cellprinter.

To characterize the cells, the library may be QC'd, and assays are run.In another option, all cell colonies are pooled, a glycerol stocklibrary is made, and selections are run on aliquots of the pooled cells.Finally, the cells are analyzed using amplicon verification or genomevalidation.

Example 7: Bulk Cell 3D Isolation, Colony Normalization, and Processingwithin a Rotating Growth Vial

Editing Bulk Cell Culture:

This protocol describes a standard bulk culture protocol using alginateas the solidifying agent. Alginate both solidifies and re-liquifies at atemperature appropriate for enriching for nucleic acid-guided nucleaseediting of bacterial and yeast cells by isolation, growth, editing, andnormalization. This protocol was used to leverage the inducible systemfor both the nuclease and gRNAs to allow for a phenotypic difference incolonies. Alginate (Alginate, A1112 Sigma-Aldrich (St. Louis, Mo.),Alginic acid sodium salt from brown algae, low viscosity).

Solutions:

TABLE 4 LB Alginate 1 L 500 ml 250 ml units notes LB 25 12.5 6.25 g LBMiller version of LB powder Broth powder (Teknova Cat. No. L9135) DI H₂O1000 500 250 ml Alginate 20 10 5 g Alginic acid sodium salt from brownalgae, low viscosity (A1112 Sigma); 2% final conc

LB and DI H₂O in desired quantities as listed in Table 4 were combinedin a flask. A stir bar was added to the flask and the alginate was addedslowly while the LB/alginate mixture was stirred on a stir plate. TheLB/alginate mixture was then sterilized by autoclavation using standardconditions (e.g., 121° C., 20 min, liquid cycle). After autoclavation,the solution was immediately cooled on ice. Before using the LB/alginatesolution, cells and desired antibiotics were added to the appropriateconcentration.

TABLE 5 LB Alginate, composition for arabinose induction (1% final conc)1 L 500 ml 250 ml units notes LB 25 12.5 6.25 g LB Miller version of LBpowder Broth powder (Teknova Cat. No. L9135) DI H₂O 950 475 237.5 mlalginate 20 10 5 g Alginic acid sodium salt from brown algae, lowviscosity (A1112 Sigma); 2% final conc 20% 50 25 12.5 ml From 20%Arabinose arabi- Solution, 1 Liter, nose Sterile. (Teknova Cat. No.A2100); 1% final conc, to be added after autoclavation, just before use

LB and DI H₂O in desired quantities as listed in Table 5 were combinedin a bottle. A stir bar was added to the bottle and the alginate wasadded slowly while the LB/alginate mixture was stirred on a stir plate.The LB/alginate mixture was then sterilized by autoclavation usingstandard conditions (e.g., 121° C., 20 min, liquid cycle). Afterautoclavation, the solution was immediately cooled on ice. Before usingthe LB/alginate solution, cells and desired antibiotics were added tothe appropriate concentration, and 1 ml of 20% arabinose also was addedto 19 ml of the LB alginate solution to obtain a 1% arabinose finalconcentration. Next, calcium chloride (1M) solution was prepared, usingcalcium chloride dihydrate, MW=147.01 g/mol, and this calcium chloridesolution was filter sterilized. Also, a 1M sodium citrate solution wasprepared, using sodium citrate tribasic dihydrate, MW=294.10 g/mol,which was also filter sterilized.

Editing was performed following the above protocols to make LB Alginate(25 ml per sample) and LB Alginate+1% arabinose (25 ml per sample). 10ml of alginate+1% arabinose solution was added to each 50 ml conicaltube. The conical tubes were kept at 30° C., to be ready for use aftertransformation protocol was complete. Transformation was performed using500 ng of the nucleic acid assembly (vector+editing cassette library)into ec83 (recombineering competent cells) using the Nepageneelectroporator settings for E. coli. The cells were allowed to recoverin 3000 μl of SOB in 15 ml conical tubes while shaking at 30° C. for 3hours. After 3 hours, the alginate tubes and the transformation tubeswere removed from the 30° C. incubator, and 250 μl of cells was added toeach tube with the 25 ml of Alginate solution (1:10). The Alginatesolution was solidified by slowly transferring 20 ml of thealginate+cells solution into 30 ml of 100 mM CaCl₂ solution. Thealginate slurry was then centrifuged for 10 min at 4000×g. Thesupernatant was decanted, and the bulk gel was incubated at 30° C. for 9hours. After the 9-hour 30° C. incubation, the temperature was shiftedto 42° C. for 2 hours for induction of editing.

After editing, the temperature was shifted back to 30° C. for growthovernight. To re-liquefy (dissolve) the alginate, 40 ml DI water wasadded to each conical tube, and 10 ml of 1M sodium citrate was added.The tubes were then shaken at 30° C. for 30-45 minutes. FIG. 6 is aphotograph of E. coli cells expressing green fluorescent protein in 2.0%alginate and medium that has been solidified showing isolated colonies(left) and a photograph of E. coli cells expressing green fluorescentprotein in 2.0% alginate and medium after the medium has beenre-liquified (right). For singleplex recovery, the libraries wererecovered by diluting the cells and plating the cells on selective mediaplates. Various dilutions were plated and plates also were spotted toget colony counts. The cells were grown on the selective medium for12-24 hours, and colonies were picked into a 96-well plate with eachwell containing 750 ml LB. The picked colonies were grown for 24 hours,and each sample was prepped for DNA extraction and next-gen sequencing.For amplicon recovery, the cells were spun at 5,000×g for 10 minutes.The supernatant was removed and the cells were resuspended in 500 μl of0.8 NaCl. A Zyppy™ Plasmid Miniprep kit (Zymo Research, Bath, UK) wasused to extract the plasmid DNA from the library, and the samples wereprepped for PCR of the inserts, and for assaying the amplicons vianext-gen sequencing.

FIGS. 7A-7C are a depiction of an experiment performed to demonstratethat normalization is achieved in bulk culture, which compares thequantity of wildtype (inert) plasmid and editing plasmid (GalK) in bulkgel versus liquid cell culture (see Example 9 below). In a first step(shown in FIG. 7A), the wildtype plasmid was used to transform an E.coli cell line, and separately, an editing plasmid was used to transformthe E. coli cell line. Once transformed, pools of the transformed cellswere combined in the following ratios: 50:50, 10:1, and 1:10 (wildtypeto editing cells, respectively) and dispensed between both bulk cultureand liquid culture, where six replicates were prepared for each.Controls included 100% wildtype and 100% editing cells, and standardplating controls. In FIG. 7B, the bulk and liquid cultures (experimentaland controls) were grown at 30° C. for 6 hours, 42° C. for 2 hours, andat 30° C. overnight. Next, live cells were recovered from each culture(e.g., six experimental cultures and controls for each of the bulk andliquid cultures). FIG. 7C depicts plasmid extraction and isolation ofthe cells recovered from the bulk gel cultures and from the liquidcultures (shown) as well as the controls (not shown). Phenotypicassessment was used to determine whether normalization takes place inthe bulk gel culture. The phenotypic read out comprised red/whitescreening on MacConkey agar. The results obtained demonstrated cellsedited in bulk gel match most closely the loaded ratio of the 50:50 mixof cells edited at 25% in alginate and 7% liquid and on a plate. FIG. 7Ddepicts the bulk gel process with recursive cell editing.

FIG. 7E is a photograph of E. coli cells expressing green fluorescentprotein in 2.0% alginate and medium that has been solidified showingsingulated colonies (left) and a photograph of E. coli cells expressinggreen fluorescent protein in 2.0% alginate and medium after the mediumhas been re-liquified.

FIG. 7F depicts the workflow for bulk alginate isolation, growth,induction, editing, and normalization in a rotating growth vial (asshown in FIG. 4A and described above), which can be used in amulti-module cell editing instrument (as shown in FIG. 3A and describedabove). In a first step, 10 ml LB medium comprising alginate was addedto the rotating growth vial, which already contained the transformedcells to be edited. In addition to the alginate, the medium alsocomprises antibiotics to select for the cells that have been properlytransformed. The medium was then solidified by slowly adding 1.5 ml of1M CaCL₂ to the LB alginate cell culture in the rotating growth vial.The cells were allowed to grow for 6 hours at 30° C. to establish cellcolonies, 2 hours at 42° C. (which induces editing), then overnight at30° C. to normalize the edited and unedited cell colonies. Afternormalization, the solidified LB alginate medium was liquified by adding10 ml 1 M sodium citrate to the solidified medium, and the liquifiednormalized cell culture was filtered in a filtration module allowing forbuffer exchange, cell concentration, and, if desired, rendering thecells electrocompetent for an additional round of editing. Liquificationdisperses all cells throughout the culture. Alternatively, the cellswere diluted and plated, and slow-growing colonies were “cherry picked”(e.g., small colonies were picked and larger colonies were not), toselect for edited cells.

Example 8: Standard Plating for Comparison to Bulk Culture or Solid WallIsolation

This protocol describes a standard plating protocol for enriching fornucleic acid-guided nuclease editing of bacterial cells by isolation,growth, editing, and normalization. This protocol was used to leveragethe inducible system for both the nuclease and gRNAs to allow for aphenotypic difference in colonies. From the resulting agar plates, it ispossible to select edited cells with a high degree (˜80%) of confidence.Though clearly this protocol can be employed for enriching for editedcells, in the experiments described herein this “standard platingprotocol” of “SPP” was used to compare efficiencies of isolation,editing, and normalization with the bulk cell culture. The protocols forliquid cell culture described in Example 7 were used for the samepurpose.

Materials:

Outside of standard molecular biology tools, the following will benecessary:

TABLE 6 Product Vendor SOB Teknova LB Teknova LB agar plate with Teknovachloramphenicol/carbenicillin and 1% arabinose

Protocol:

Inputs for this protocol are frozen electrocompetent cells and purifiednucleic acid assembly product. Immediately after electroporation, thecell/DNA mixture was transferred to a culture tube containing 2.7 mL ofSOB medium. Preparing 2.7 mL aliquots in 14 mL culture tubes prior toelectroporation allowed for a faster recovery of cells from theelectroporation cuvette; the final volume of the recovery was 3 mL. Allculture tubes were placed into a shaking incubator set to 250 RPM and30° C. for three hours. While the cultures were recovering, thenecessary number of LB agar plates with chloramphenicol andcarbenicillin+1% arabinose were removed from the refrigerator and warmedto room temperature. Multiple dilutions were used for each plating so asto have countable and isolated colonies on the plates. Platingsuggestions:

TABLE 7 Dilution(s) Sequencing type suggested Volume to plate SinglePlex10⁻¹ through 10⁻³ 300 uL Amplicon None 300 uL (= 1/10^(th) recovery)

After three hours, the culture tubes were removed from the shakingincubator. First, plating for amplicon sequencing was performed byfollowing the above table. Plating beads were used to evenly distributethe culture over the agar. The beads were removed from the plate theplate was allowed to dry uncovered in a flow hood. While the plates weredrying, the remaining culture was used to perform serial dilutions,where the standard dilutions were 50 μL of culture into 450 uL ofsterile, 0.8% NaCl. The plate/tubes used for these dilutions (as well asthe original culture) were maintained at 4° C. in case additionaldilutions were needed to be performed based on colony counts. Platingfor SinglePlex was performed according to the Table 7. Additional orfewer dilutions may be used based on library/competent cell knowledge.The cultures were evenly spread across the agar using sterile, platingbeads. The beads were then removed from the plate and the plates wereallowed to dry uncovered in the flow hood. While the plates were drying,an incubator was programmed according to the following settings: 30° C.for 9 hours→42° C. for 2 hours→30° C. for 9 hours. The agar plates wereplaced in the pre-set incubator, and after the temperature cycling wascomplete (˜21 hours), the agar plates were removed from the incubator.If induction of editing has been successful, size differences in thecolonies will be visible.

Example 9: Liquid Cell Culture Procedure for Comparison to Bulk Culture

Liquid culture process for control: The editing cassette libraries weretransformed via electroporation into specific strains of E. coliexpressing Mad7 (nuclease) and Lambda Red (recombination) proteins.Transformation of process control vectors—alongside the editing cassettelibraries—is essential to calculate the transformation efficiency andediting efficiency (sgRNA efficiency). Immediately post-transformation,the electroporated cells were transferred to medium for recovery.

TABLE 8 Summary of Related QC Assays/M-Tools QC Assay What is beingModule Description measured Input Output Transforma- Live/dead #startinglive/dead # live/dead tion flow after cells, # stain, cells cells,(supercoiled transforma- live cells after selective/non- plasmids) tionwith plasmid transf. selective conditions Transforma- Live/dead#starting live/dead # live/dead tion flow after cells, # stain, cellscells, (direct to transforma- live cells after selective/non- test) tionwith plasmid transf. selective conditions

Following electroporation and recovery, cells from these process controltransformations were spread on LB agar plates with the appropriateantibiotics. After overnight growth on plates, cells are scraped andthen plated on the selective MacConkey phenotypic agar plates for thesugar edits that are tested: Xylose, Galactose, or Lactose, or scrapedand replated on LB agar to determine clonality of the individual cellsfrom plates. In more detail, after a 3-hour incubation (recovery), theculture tubes were removed from the shaking incubator. While the culturetubes were incubating, 250 mL baffled shake flasks were prepared with 25mL of LB+100 ug/mL carbenicillin and 25 ug/mL chloramphenicol and 1%arabinose. After incubation, 250 μL of undiluted culture from eachtransformation was transferred into the prepared 250 mL shake flasks. Anincubator was set to the following temperature settings: 30° C. for 9hours→42° C. for 2 hours→30° C. for 9 hours. This temperature regime wasused to allow for additional recovery during the first nine hoursfollowed by induction of the nuclease during the two-hour step. Thelambda (recombineering system) induction was triggered by arabinose inthe medium. The flasks were incubated/shaken at 250 RPM. After thetemperature cycling was complete (˜21 hours), the flasks were removedfrom the incubator/shaker.

Serial dilutions of each culture were prepared with 0.8% NaCl, where thestandard dilutions were 50 μL of culture into 450 μL of sterile, 0.8%NaCl, and dilutions of 10⁻⁵ to 10⁻⁷ were made to produce isolatedcolonies. 300 uL of each dilution of each culture was plated onto LBagar plates with standard concentrations of chloramphenicol andcarbenicillin. Arabinose was not used in the agar plates as all editingshould have occurred in the incubation/shaking process. The plates wereplaced in a 30° C. incubator for overnight growth where colonies formedovernight and were picked for singleplex next-gen sequencing thefollowing day using 250 μL of culture as the input for a plasmidextraction protocol.

Example 10: Results

FIGS. 8A, 8B, and 8C show the results of the editing rates and clonalityresulting from editing experiments performed with liquid cell cultureemploying no isolation or normalization, but employing inducibleediting; bulk cell gel culture, employing isolation, inducible editing,and normalization; solid agar plating (SPP) employing isolation,inducible editing, and normalization; solid agar plating (SPP-Cherry)employing isolation, inducible editing, and cherry picking; and solidagar plating (SPP) employing only isolation and inducible editing andsimply scraping the colonies from the plate and re-plating.

FIG. 8A shows that liquid culture results in a very low rate of observedediting, at about 1-2%; the standard plating procedure (SPP) results inan approximate 75% rate of observed editing; the bulk alginate cellculture protocol results in an approximate 50% rate of observed editing;the standard plating procedure plus cherry picking (SPP-cherry) (e.g.,manual picking of only small colonies from the plated cells, where thepresumption is that small colonies represent colonies of cells that havebeen edited) protocol results in an approximate 95% rate of observedediting; and the standard plating procedure (SPP) without normalizationor cherry picking results in an approximate 8% of observed editing.Thus, it is clear that SPP+cherry picking produces the highest rate ofobserved editing. In addition, SPP without cherry picking—but includingisolation, induced editing, and normalization—results in a high (75%)rate of observed editing, and the easily-automatable bulk gel cellculture process resulted in an approximate 50% rate of observed editing.

FIG. 8B provides the observed clonality for the standard platingprocedure (SPP), the standard plating procedure+cherry picking (SPPcherry), the standard plating procedure+scraping the plate comprisingthe colonies where editing has been induced (but also comprisingunedited cells), and for the bulk procedure. The first column gives thefraction of colonies examined with more than half the reads being callededits. The second column gives the fraction of colonies that have morethan 90% of the reads being called edit reads. The higher fraction hereshows how complete the edits are if there are some colonies examinedbetween the 50% and 90% cut offs that demonstrate that not all of thecells in the colony that is being picked are edited. That is when onecell hits the plate and begins growing into a clonal colony for, e.g.,˜100 cells—then editing is induced—some of the cells are edited but notall, and the cells that are not edited cause this incomplete editing inthe colony (e.g., less than 100% clonality). The third column providesthe number of unique edits for the colonies in the >50% clonal colonies.Note that SPP-cherry provides the highest clonality and number of uniqueedits, but that the bulk gel cell culture provides good clonality (44/95at >50%) and a high proportion of the clonal colonies consist of uniqueedits (42/44).

Finally, FIG. 8C provides a graph of the data in FIG. 8B. This graphindicates the extent of incomplete editing.

Example 11: Solid Wall Embodiment

FIG. 9A is a photograph of one embodiment of a solid wall devicecomprising microwells for isolating cells. As can be seen from thephoto, the solid wall device is approximately 2 inches (˜47 mm) indiameter. The solid device seen in this photograph is essentially aperforated disk of 316 stainless steel, where the perforations form thewalls of the microwells, and a filter or membrane is used to form thebottom of the microwells. Use of a filter or membrane (such as a 0.22μPVDF Duropore™ woven membrane filter) allows for medium and/or nutrientsto enter the microwells but prevents the cells from flowing down and outof the microwells. Filter or membrane members that may be used in thesolid wall isolation/growth/editing/normalization devices and modulesare those that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 μm or larger. Indeed,the pore sizes useful in the cell concentration device/module includefilters with sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm,0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm,0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm,0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm,0.49 μm, 0.50 μm and larger. The filters may be fabricated from anysuitable material including cellulose mixed ester (cellulose nitrate andacetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glassfiber.

In the photograph shown in FIG. 9A, the perforations are approximately152 nM in diameter, resulting in the microwells having a volume ofapproximately 2.5 nL, with a total of approximately 30,000 wells. Thedistance between the microwells is approximately 279 nMcenter-to-center. Though here the microwells have a volume ofapproximately 2.5 nL, the volume of the microwells may be from 1 to 25nL, or preferably from 2 to 10 nL, and even more preferably from 2 to 4nL. The preferred size/volume of the microwells will depend of cell type(e.g., bacterial, yeast, mammalian). The perforated disk shown here ismade of 316 stainless steel; however other bio-compatible metals andmaterials may be used. The solid wall device may be disposable or it maybe reusable. The solid wall device shown in FIG. 9A is round, but can beof any shape, for example, square, rectangular, oval, etc. (See, e.g.,the rectangular perforated member in the SWIIN depicted in FIGS. 5A-5I.)Round solid wall devices are useful if petri dishes are used to supplythe solid wall device with nutrients via solid medium. The filters usedto form the bottom of the wells of the solid wall device include 0.22μPVDF Duropore™ woven membrane filters. Further, though a 2-inch (˜47 mm)diameter solid wall device is shown, the solid wall devices may besmaller or larger as desired and the configuration of the solid walldevice will depend on how nutrients are supplied to the solid walldevice, and how media exchange is performed (see, e.g., the solid walldevice described in relation to Example 18, FIGS. 14A and 14B).

FIGS. 9B-9D are photographs of E. coli cells isolated via Poissondistribution in microwells in a solid wall device with a membrane bottomat low, medium and high magnification, respectively. FIG. 9B showsdigital growth at low magnification where the darker microwells aremicrowells where cells are growing. FIG. 9C is a top view of microwellsin a solid wall device where the darker microwells are microwells wherecells are growing. FIG. 9D is a photograph of microwells where themembrane (e.g., the permeable membrane that forms the bottom of themicrowells) has been removed, where unpatterned (smooth) microwells aremicrowells where cells are not growing, and microwells with irregularpigment/patterned are microwells where cells are growing, and, in thisphotograph, have filled the microwells in which they are growing. Inthese photographs, a 0.2 μm filter (membrane) was pressed against theperforated metal sold wall device such as the round solid wall devicedepicted in FIG. 9A. The perforated metal solid wall device formed thewalls of the microwells, and the 0.2 μm filter formed the bottom of themicrowells. To load the solid wall device, the E. coli cells were pulledinto the microwells using a vacuum. The solid wall device+filter wasthen placed on an LB agar plate membrane-side down, and the cells weregrown overnight at 30° C., then two days at room temperature. Themembrane was removed and the bottomless microwells were photographed bylight microscopy. Note the ease with which different selective media canbe used to select for certain cell phenotypes; that is, one need onlytransfer the solid wall device+filter to a different plate or petri dishcomprising a desired selective medium.

Example 12: Isolation and Culture of E. coli in a Solid Wall Device

Electrocompetent E. coli cells were transformed with a cloned library,an isothermal assembled library, or a process control sgRNA plasmid(escapee surrogate) as described in Example 3 above. The E. coli straincarried the appropriate endonuclease and lambda red components andediting induction system (e.g., on an engine plasmid or integrated intothe bacterial genome or a combination). Transformations routinely used150 ng of plasmid DNA (or Gibson Assembly reactions) with 150 ng of pLsgRNA backbone DNA. Following electroporation, the cells were allowed torecover in 3 ml SOB and incubated at 30° C. with shaking for 3 hours. Inparallel with processing samples through the solid wall device, sampleswere also processed with the solid plating protocol (see Example 8above), so as to compare “normalization” in the sold wall device withthe standard benchtop process. Immediately before cells the cells wereintroduced to the permeable-bottom solid wall device, the 0.2 μm filterforming the bottom of the microwells was treated with a 0.1% TWEENsolution to effect proper spreading/distribution of the cells into themicrowells of the solid wall device. The filters were placed into aSwinnex Filter Holder (47 mm, Millipore®, SX0004700) and 3 ml of asolution with 0.85% NaCl, and 0.1% TWEEN was pulled through the solidwall device and filter through using a vacuum. Different TWEENconcentrations were evaluated, and it was determined that for a 47 mmdiameter solid wall device with a 0.2 M filter forming the bottom of themicrowells, a pre-treatment of the solid wall device+filter with 0.1%TWEEN was preferred (data not shown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 ml volume of the diluted cells was processed through theTWEEN-treated solid wall device and filter, again using a vacuum. Thenumber of successfully transformed cells was expected to beapproximately 1.0E+06 to 1.0E+08, with the goal of loading approximately10,000 transformed cells into the current 47 mm permeable-bottom solidwall device (having ˜30,000 wells). Serial dilutions of 10⁻¹, 10⁻², and10⁻³ were prepared, then 100 μL volumes of each of these dilutions werecombined with 3 ml 0.85% NaCl, and the samples were loaded onto solidwall devices. Each permeable-bottom solid wall device was then removedfrom the Swinnex filter holder and transferred to an LB agar platecontaining carbenicillin (100 μg/ml), chloramphenicol (25 μg/ml) andarabinose (1% final concentration). The solid wall devices were placedmetal side “up,” so that the permeable-bottom membrane was touching thesurface of the agar such that the nutrients from the plate could travelup through the filter “bottom” of the wells. The solid wall devices onthe LB agar plates were incubated for 9 hours at 30° C., at 42° C. for 2hours, then returned to incubation at 30° C., for 12-16 hour, and, inanother experiment for 36-40 hours.

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 ml of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereallowed cells to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 ml conical centrifuge tube. The OD600 of thecell suspension was measured and at this point, the cells can beprocessed in different ways depending on the purpose of the study. Forexample, if the plasmids or libraries are designed to target a sugarmetabolism pathway gene such as galK, then the resuspended cells can bespread onto MacConkey agar plates containing galactose (1% finalconcentration) and the appropriate antibiotics. On this differentialmedium, colonies that are the result of successfully-edited cells areexpected to be phenotypically white in color, whereas unedited coloniesare red in color. This red/white phenotype can then be used to assessthe percentage of edited cells and the extent of normalization of editedand unedited cells. The results of one experiment are shown below inTable 9. In all replicates, the transformed cells were allowed to growin the solid wall devices for 9 hours at 30° C., 2 hours at 42° C., andovernight at 30° C.

TABLE 9 Dilution Red White Tween? counted colonies colonies % edit Notween 10⁻⁴ 72 5 6% No tween 10⁻⁴ 89 3 3% No tween 10⁻³ 64 5 7%Pre-treatment tween 10⁻⁴ 71 5 7% Pre-treatment tween 10⁻³ 443 29 6%Pre-treatment tween 10⁻³ 149 12 7% Pre-treatment tween 10⁻³ 83 21 20% Pre-treatment tween 10⁻² 318 112 26%  Pre-treatment tween + tween in10⁻³ 163 25 13%  cell loading buffer Pre-treatment tween + tween in 10⁻⁴132 10 7% cell loading buffer Pre-treatment tween + tween in 10⁻⁴ 31 923%  cell loading buffer Pre-treatment tween + tween in 10⁻³ 147 1810.9%   cell loading buffer Pre-treatment tween + tween in 10⁻² 720 15017%  cell loading buffer Pre-treatment tween + tween in 10⁻³ 55 15 21% cell loading buffer

The solid wall device+filter was pre-treated with 0.1% TWEEN to assistin dispersing the cells on the solid wall device, and a 10⁻³ dilution ofthe transformed cells was introduced to the solid wall device. FIG. 9Eis a graph showing the extent of normalization of cells (% edited cells)for different dilutions of transformed cells, and no treatment withTWEEN vs. pre-treatment with TWEEN vs. pre-treatment with TWEEN+TWEEN inthe buffer when loading the cells into the microwells of the solid walldevice. A standard plating protocol (SPP) was conducted in parallel withthe solid wall isolation experiments as a benchmark (first bar on theleft in the graph). Note that the percentage of edits for the standardplating protocol was approximately 27.5%, and the percentage of editsfor two replicates of the 10⁻³ dilution of cells with pre-treatment withTWEEN was approximately 20% and 26%, respectively.

Example 13: Isolation of Yeast Colonies in a Solid Wall Device

Electrocompetent yeast cells were transformed with a cloned library, anisothermal assembled library, or a process control sgRNA plasmid(escapee surrogate) as described in Example 3 above. ElectrocompetentSaccharomyces cerevisiae cells were prepared as follows: The afternoonbefore transformation was to occur, 10 mL of YPAD was inoculated withthe selected S. cerevisiae strain. The culture was shaken at 250 RPM and30° C. overnight. The next day, 100 mL of YPAD was added to a 250-mLbaffled flask and inoculated with the overnight culture (around 2 mL ofovernight culture) until the OD600 reading reached 0.3+/−0.05. Theculture was placed in the 30° C. incubator shaking at 250 RPM andallowed to grow for 4-5 hours, with the OD checked every hour. When theculture reached an OD600 of approximately 1.5, 50 mL volumes were pouredinto two 50-mL conical vials, then centrifuged at 4300 RPM for 2 minutesat room temperature. The supernatant was removed from all 50 ml conicaltubes, while avoiding disturbing the cell pellet. 50 mL of a LithiumAcetate/Dithiothreitol solution was added to each conical tube and thepellet was gently resuspended. Both suspensions were transferred to a250 mL flask and placed in the shaker; then shaken at 30° C. and 200 RPMfor 30 minutes. After incubation was complete, the suspension wastransferred to two 50-mL conical vials. The suspensions then werecentrifuged at 4300 RPM for 3 minutes, then the supernatant wasdiscarded. Following the Lithium Acetate/Dithiothreitol treatment step,cold liquids were used and the cells were kept on ice untilelectroporation.

50 mL of 1 M sorbitol was added and the pellet was resuspended, thencentrifuged at 4300 RPM, 3 minutes, 4° C., after which the supernatantwas discarded. The 1M sorbitol wash was repeated twice for a total ofthree washes. 50 μL of 1 M sorbitol was added to one pellet, cells wereresuspended, then transferred to the other tube to suspend the secondpellet. The volume of the cell suspension was measured and brought to 1mL with cold 1 M sorbitol. At this point the cells were electrocompetentand could be transformed with a cloned library, an isothermal assembledlibrary, or process control sgRNA plasmids. In brief, a required numberof 2-mm gap electroporation cuvettes were prepared by labeling thecuvettes and then chilling on ice. The appropriate plasmid—or DNAmixture—was added to each corresponding cuvette and placed back on ice.100 μL of electrocompetent cells was transferred to each labelledcuvette, and each sample was electroporated using appropriateelectroporator conditions. 900 uL of room temperature YPAD Sorbitolmedia was then added to each cuvette. The cell suspension wastransferred to a 14 ml culture tube and then shaken at 30° C., 250 RPMfor 3 hours. After a 3 hr recovery, 9 ml of YPAD containing theappropriate antibiotic, e.g., geneticin or Hygromycin B, was added. Atthis point the transformed cells were processed in parallel in the solidwall device and the standard plating protocol (see Example 8 above), soas to compare “normalization” in the sold wall device with the standardbenchtop process. FIG. 9F is a photograph of a solid wall device with apermeable bottom on agar, on which yeast cells have been isolated andgrown into clonal colonies. FIG. 9G presents photographs of yeast colonygrowth at various time points. Immediately before cells the cells wereintroduced to the permeable-bottom solid wall device, the 0.45 μM filterforming the bottom of the microwells was treated with a 0.1% TWEENsolution to effect proper spreading/distribution of the cells into themicrowells of the solid wall device. The filters were placed into aSwinnex Filter Holder (47 mm, Millipore®, SX0004700) and 3 ml of asolution with 0.85% NaCl and 0.1% TWEEN was pulled through the solidwall device and filter through using a vacuum. Different TWEENconcentrations were evaluated, and it was determined that for a 47 mmdiameter solid wall device with a 0.45 M filter forming the bottom ofthe microwells, a pre-treatment of the solid wall device+filter with0.1% TWEEN was preferred (data not shown).

After the 3-hour recovery in SOB, the transformed cells were diluted anda 3 ml volume of the diluted cells was processed through theTWEEN-treated solid wall device and filter, again using a vacuum. Thenumber of successfully transformed cells was expected to beapproximately 1.0E+06 to 1.0E+08, with the goal of loading approximately10,000 transformed cells into the current 47 mm permeable-bottom solidwall device (having ˜30,000 wells). Serial dilutions of 10⁻¹, 10⁻², and10⁻³ were prepared, then 100 μL volumes of each of these dilutions werecombined with 3 ml 0.85% NaCl, and the samples were loaded onto solidwall devices. Each permeable-bottom solid wall device was then removedfrom the Swinnex filter holder and transferred to an LB agar platecontaining carbenicillin (100 μg/ml), chloramphenicol (25 μg/ml) andarabinose (1% final concentration). The solid wall devices were placedmetal side “up,” so that the permeable-bottom membrane was touching thesurface of the agar such that the nutrients from the plate could travelup through the filter “bottom” of the wells. The solid wall devices onthe YPD agar plates were incubated for 2-3 days at 30° C.

At the end of the incubation the perforated disks and filters (stillassembled) were removed from the supporting nutrient source (in thiscase an agar plate) and were photographed with a focused,“transilluminating” light source so that the number and distribution ofloaded microwells on the solid wall device could be assessed (data notshown). To retrieve cells from the permeable-bottom solid wall device,the filter was transferred to a labeled sterile 100 mm petri dish whichcontained 15 ml of sterile 0.85% NaCl, then the petri dish was placed ina shaking incubator set to 30° C./80 RPM to gently remove the cells fromthe filter and resuspend the cells in the 0.85% NaCl. The cells wereallowed cells to shake for 15 minutes, then were transferred to asterile tube, e.g., a 50 ml conical centrifuge tube. The OD600 of thecell suspension was measured; at this point the cells can be processedin different ways depending on the purpose of the study. For example, ifan ADE2 stop codon mutagenesis library is used, successfully-editedcells should result in colonies with a red color phenotype when theresuspended cells are spread onto YPD agar plates and allowed to growfor 4-7 days. This phenotypic difference allows for a quantification ofpercentage of edited cells and the extent of normalization of edited andunedited cells.

Example 14: Isolation, Growth and Editing of E. coli in 200K SWIIN

Singleplex automated genomic editing using MAD7 nuclease, a library with94 different edits in a single gene (yagP) and employing a 200K SWIINmodule such as that exemplified in FIGS. 5A-5I was successfullyperformed. The engine vector used had MAD7 under the control of the pLinducible promoter and the λRed system under the control of a pBADpromoter), and the editing vector used comprised an editing cassette(gRNA and donor DNA) being under the control of the pL induciblepromoter. Two SWIIN workflows were compared, and further werebenchmarked against the standard plating protocol (see Example 8). TheSWIIN protocols different from one another that in one set of replicatesLB medium containing arabinose was used to distribute the cells in theSWIIN (arabinose was used to induce the λ Red recombineering system(which allows for repair of double-strand breaks in E. coli that arecreated during editing), and in the other set of replicates SOB mediumwithout arabinose was used to distribute the cells in the SWIIN and forinitial growth, with medium exchange performed to replace the SOB mediumwithout arabinose with SOB medium with arabinose. Approximately 70Kcells were loaded into the 200K SWIIN.

In all protocols (standard plating, LB-SWIIN, and SOB-SWIIN), the cellswere allowed to grow at 30° C. for 9 hours and editing was induced byraising the temperature to 42° C. for 2.5 hours, then the temperaturewas returned to 30° C. and the cells were grown overnight. The resultsof this experiment are shown in FIG. 10 and in Table 10 below. Note thatsimilar editing performance was observed with the four replicates of thetwo SWIIN workflows, indicating that the performance of SWIIN platingwith and without arabinose in the initial medium is similar. Editingpercentage in the standard plating protocol was approximately 77%, inbulk liquid was approximately 67%, and for the SWIIN replicates rangedfrom approximately 63% to 71%. Note that the percentage of unique editcassettes divided by the total number of edit cassettes was similar foreach protocol.

TABLE 10 Stan- SWIIN SWIIN dard SWIIN SWIIN SOB then SOB then Plat-LB/Ara LB/Ara SOB/Ara SOB/Ara ing Rep. A Rep. B Rep. A Rep. B 40006 editcalls/ 0.777 0.633 0.719 0.663 0.695 identified wells Unique editcassettes/ 0.49 0.49 0.43 0.50 0.51 total edit cassettes

Example 15: Protocol Flow from mTFF to FTEP to SWIIN

mTFF module, E. coli workflow: Approximately 20 ml of E. coli wastransferred from a rotating growth vial (RGV) in a cell growth module.Specially, the E. coli was EC83, an E. coli MG1655 strain comprising anengine vector coding for the λRed recombineering system and a MAD7coding sequence. In the RGV, the EC83 was grown in LB growth medium toan OD600 ˜0.6 and having a conductivity of ˜16,500 μS/cm. In the mTFFthe cells were washed with a low-conductivity solution (10% glycerol)and concentrated in a small volume (approximately 0.80 ml) in the samelow-conductivity solution. Both the input and output cell counts weredetermined by plating on solid media. The cell input was approximately3.1E+09 and the cell output was approximately 2.3E+09. The output of themTFF was used as input for the flow-through electroporation (FTEP)module.

FTEP Module, E. coli Workflow:

Approximately 500 μl of the concentrated EC83 cells in 10%glycerol—e.g., an aliquot of the output of the mTFF—was combined with100 μl of the assembled editing vector (see, e.g., FIG. 11B) and a TWEENsolution (see Example 12 above). The cells and DNA were mixed and passedthrough the FTEP at a very high field strength in a high resistancesolution. After transformation in the FTEP, the cells were transferredback to the cell growth module into a fresh RGV containing 3 ml SOB withchloramphenicol. The cells were incubated at 30° C. for 1 hour. Theinput CFU was approximately 2.3E+09 and the output CFU was approximately9.8E+08 survival and 8.5E+05 uptake.

SWIIN Module, E. coli Workflow:

Subsequent to the 1 hour recovery in the cell growth module, the cellswere combined with a PBS/TWEEN solution and approximately 0.35 ml wasloaded onto the SWIIN. Once cells were loaded and growth medium (was LBwith 1% arabinose, 25 ug/ml chloramphenicol and 100 μg/ml carbenicillin)was added to the permeate chamber of the SWIIN, the SWIIN was placed ina programmable incubator for the induction and editing stages. Thescript for the SWIIN protocol was set for a 9 hour incubation at 30° C.,a 2.5 hour incubation at 42° C., then 9 additional hours at 30° C. TheCFU input into the SWIIN was approximately 7.5E+04 and the CFU outputwas approximately 6.5E+06 in a 7.0 ml volume.

Amplicon and Singleplex Sequencing:

Cells were recovered from the SWIIN with a PBS solution, and 100 μl ofthe undiluted cell suspension was spread on an LB chlor/carb agar plateand incubated overnight at 30° C. This plate was then “scraped” and usedas the amplicon DNA input. Dilutions were also prepared from thePBS/cell suspension and then plated on an LB chlor/carb agar plate andincubated overnight at 30° C.; isolated colonies were selected and usedfor singleplex sequencing.

Example 16: Loading and Performing Editing on a SWIIN

FIGS. 11A-11C are simplified overviews of various parameters for loadingcells onto a SWIIN module, performing editing, and removing orrecovering the edited cells from the SWIIN module. The steps of FIGS.11A-11C correspond to the steps listed in Tables 1-3, with the exceptionthat step 1 (loading the SWIIN module into the automated multi-modulecell processing instrument) and step 32 (unloading the SWIIN from theautomated multi-module cell processing instrument) are not representedon FIGS. 11A-11C. FIG. 11A begins with step 2, where 10 mL of PBS/0.01%Tween80 was transferred from a reagent cartridge to permeatereservoir 1. At this initial step, retentate reservoir 1, retentatereservoir 2, and permeate reservoir 1 were under positive pressure, flowsensor 1 detected a high flow rate and flow sensor 2 detected a low flowrate. At step 3, additional PBS/0.01% Tween80 was loaded into thepermeate channel and a bubble flush was performed. At step 4, morePBS/0.01% Tween80 was loaded into the permeate channel to fill thepermeate channel. Step 4 ended with a flow meter trigger, and flowsensor 1 returned to baseline once the permeate channel was filled withliquid.

At step 5, a vacuum was applied at retentate reservoirs 1 and 2, and theretentate channel was flooded. Once the retentate channel was flooded(and there was minimal fluid remaining in the permeate reservoirs), thenegative pressure (vacuum) was removed. At step 6, negative pressure wasapplied to retentate reservoir 2, thereby sweeping all liquid toretentate reservoir 2. At step 7, the liquid in retentate reservoirs 1and 2 was removed by, e.g., an air displacement pipette. At step 8, 9.5mL of PBS/0.01% Tween80 was transferred from the reagent cartridge toretentate reservoir 1, and at step 9, 0.5 mL of transformed cells weretransferred from the transformation module (the flow-throughelectroporation device) to retentate reservoir 1. At step 10, thecell-containing liquid was pipetted up and down in retentate reservoir1, and at step 11, the cell-containing liquid was pulled from retentatereservoir 1 into the retentate channel leaving minimal fluid inretentate reservoirs 1 and 2. Step 11 is sensitive to timing and wasthus controlled via air displacement pipette liquid level detection inretentate reservoir 1.

FIG. 11B begins with step 12, in which the fluid in the serpentinechannel in the retentate member was pulled through the membrane orfilter member on low vacuum; that is, negative pressure was applied toboth permeate reservoirs 1 and 2. At step 13, the fluid in theserpentine channel in the retentate member was pulled through the filtermember on high vacuum, with fluid pulled into the serpentine channel inthe permeate member and then into permeate reservoirs 1 and 2. At step14, all fluid was swept to permeate reservoir 1 by applying positivepressure to retentate reservoirs 1 and 2 and permeate reservoir 2.Remaining liquid was aspirated out of permeate reservoirs 1 and 2 atstep 5, with the air displacement pipette accessing permeatereservoir 1. At step 16, 10 mL medium was transferred from the reagentcartridge to permeate reservoir 1, and at step 17, the medium wastransferred from permeate reservoir 1 into the permeate channel. Step 17is sensitive to timing and thus was controlled via liquid leveldetection via a air displacement pipette in retentate reservoir 1. Atthe end of step 17, some amount of liquid resided in permeate reservoir2, and at step 18, liquid (permeate) was aspirated out of permeatereservoirs 1 and 2.

Steps 19-23 are not represented in FIGS. 11A-11C. At step 19, the SWIINmodule was incubated at 30° C. with intermittent airflow and mediumrinses/exchanges, as deemed necessary. In step 20, the temperature ofthe SWIIN module was raised to 42° C., and at step 21 the SWIIN modulewas incubated for 2 hours. At step 22, the temperature of the SWIINmodule was ramped down from 42° C. to 30° C., and the SWIIN was thenincubated at 30° C. for 9 hours. During this incubation the manifoldarms of the SWIIN assembly may be unsealed and resealed to effectairflow. In addition, media rinses or exchanges may be performed.

FIG. 11C begins with step 24, where medium was pulled out of thepermeate channel into permeate reservoir 2, by applying negativepressure to permeate reservoir 2. During this time, the flow rate forflow sensor 1 and flow sensor 2 are roughly the same at 0, then flowsensor 2 spiked to 0, rebounded, then spiked to 0 again triggering flowsensor 2. Step 24 ended with fluid in permeate reservoir 2. At step 25,the liquid was aspirated out of permeate reservoir 2 by applying avacuum to permeate reservoir 2, and at step 26, 10 mL of mediumcontaining 10% glycerol was transferred from the reagent cartridge topermeate reservoir 1. At step 27 the medium/10% glycerol was pulled frompermeate reservoir 1 into the permeate channel, and at the end of thisstep, a minimal amount of fluid remains in permeate reservoir 2. At step28, the retentate channel was flooded to dislodge the cells withpositive pressure applied to permeate reservoirs 1 and 2, thus pushingfluid from the permeate reservoirs into the retentate reservoirs. Nextat step 29, all fluid was swept to retentate reservoir 2 by applying avacuum to retentate reservoir 2. Step 29 is controlled by the trigger offlow sensor 1. Step 30 involved aspirating the cell solution fromretentate reservoir 2 into a vial, and step 31 involved aspirating allliquid out of both retentate reservoirs. The final step, step 32, is notrepresented on FIG. 11C, but involved removing the SWIIN from theautomated multi-module cell processing instrument.

Example 17: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument such asthat shown in FIGS. 5A-5D. See U.S. Pat. No. 9,982,279, issued 29 May2018 and Ser. No. 10/240,167, issued 9 Apr. 2019.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via isothermal nucleic acid assembly into an “editing vector”in an isothermal nucleic acid assembly module included in the automatedinstrument. lacZ_F172 functionally knocks out the lacZ gene.“lacZ_F172*” indicates that the edit happens at the 172nd residue in thelacZ amino acid sequence. Following assembly, the product was de-saltedin the isothermal nucleic acid assembly module using AMPure beads,washed with 80% ethanol, and eluted in buffer. The assembled editingvector and recombineering-ready, electrocompetent E. coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) and allowed to recover inSOC medium containing chloramphenicol. Carbenicillin was added to themedium after 1 hour, and the cells were allowed to recover for another 2hours. After recovery, the cells were held at 4° C. until recovered bythe user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 18: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via isothermal nucleic acidassembly into an “editing vector” in an isothermal nucleic acid assemblymodule included in the automated system. Similar to the lacZ_F172 edit,the lacZ_V10 edit functionally knocks out the lacZ gene. “lacZ_V10”indicates that the edit happens at amino acid position 10 in the lacZamino acid sequence. Following assembly, the product was de-salted inthe isothermal nucleic acid assembly module using AMPure beads, washedwith 80% ethanol, and eluted in buffer. The first assembled editingvector and the recombineering-ready electrocompetent E. coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothernal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid residue, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers are performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. An automated stand-alone multi-module cell editinginstrument comprising: a housing configured to house all or some of themodules; a receptacle configured to receive cells; a receptacleconfigured to receive editing nucleic acids; a growth module for growingcells; a filtration module for concentrating and rendering cellselectrocompetent; a transformation module configured to introduce theediting nucleic acids into the cells; a singulation and editing moduleconfigured to isolate the transformed cells and allow the editingnucleic acids to edit nucleic acids in the cells wherein the singulationand editing module comprises a device for emulsion formation,comprising: a microfluidic device having an emulsion formation unitincluding a sample well configured to receive cells in aqueous medium; acarrier fluid well configured to receive a fluid that is immiscible withthe cells in aqueous medium; a collection substrate to collect aqueousdroplets formed in the immiscible fluid; a sample channel extending fromthe sample well to a channel intersection; a carrier fluid channelextending from the carrier fluid well to the channel intersection; adroplet channel extending from the channel intersection to thecollection substrate; and a pneumatic assembly having a pressure sourceand a pressure sensor, wherein the pneumatic assembly is configured (a)to apply pressure to the emulsion formation unit to drive generation ofdroplets at the channel intersection of the emulsion formation unit andcollect droplets in the collection substrate, (b) to monitor thepressure with the pressure sensor, and (c) to stop application of thepressure to the emulsion formation unit when the pressure sensor detectsa change in pressure indicative of air entering the sample channel fromthe sample well; a processor configured to operate the automatedmulti-module cell editing instrument based on user input and/orselection of a pre-programmed script; and an automated liquid handlingsystem to move liquids from the cell receptacle to the growth module,from the growth module to the filtration module, from the filtrationmodule to the transformation module, from the nucleic acid receptacle tothe transformation module, and from the transformation module to thesingulation and editing module without user intervention.
 2. Theautomated stand-alone multi-module cell editing instrument of claim 1,wherein the singulation and editing module further comprises a detectionstation downstream from the channel intersection but before thecollection substrate.
 3. The automated stand-alone multi-module cellediting instrument of claim 2, wherein the detection station comprises acamera.
 4. The automated stand-alone multi-module cell editinginstrument of claim 2, further comprising a temperature-controlledediting reservoir positioned between the channel intersection and thedetection station.
 5. The automated stand-alone multi-module cellediting instrument of claim 4, wherein the detection station detects theoptical density of cells in the aqueous droplets.
 6. The automatedstand-alone multi-module cell editing instrument of claim 5, furthercomprising a droplet sorter positioned between the detection station andthe collection substrate.
 7. The automated stand-alone multi-module cellediting instrument of claim 6, wherein the collection substratecomprises two receptacles.
 8. The automated stand-alone multi-modulecell editing instrument of claim 1, wherein the collection substratecomprises wells, and is configured to collect one droplet per well. 9.The automated stand-alone multi-module cell editing instrument of claim8, wherein the collection substrate is temperature-controlled.
 10. Theautomated stand-alone multi-module cell editing instrument of claim 9,further comprising a detection station configured to detect droplets inthe collection substrate.
 11. A method for isolating and editing cellsin the automated stand-alone multi-module cell editing instrument ofclaim 1, comprising the steps of: providing live cells in the receptacleconfigured to receive the live cells; providing editing nucleic acidsthe receptacle configured to receive editing nucleic acids; growing thelive cells in a growth module to a desired optical density to producegrown cells; filtering and rendering electrocompetent the grown cells toproduce filtered cells; transforming the filtered cells in atransformation module configured to introduce the editing nucleic acidsinto the filtered cells to produce transformed cells; generatingdroplets in the microfluidic device by providing the transformed cellsin an aqueous medium in the sample well; providing the fluid immisciblewith the cells in the aqueous medium in the carrier fluid well; flowingthe immiscible fluid from the carrier fluid well through the carrierchannel to the channel intersection; flowing the cells in aqueous mediumfrom the sample well through the sample channel to the channelintersection; generating aqueous droplets in the immiscible fluid; andcollecting the aqueous droplets in wells in the collection substrate;incubating the aqueous droplets in the collection substrate to allow theediting nucleic acids to edit the transformed cells; pooling the aqueousdroplets; and using an automated liquid handling system to 1) transferthe editing nucleic acids from receptacle configured to receive nucleicacids to the transformation module, 2) transfer the live cells from thereceptacle configured to receive the live cells to the growth module, 3)transfer the grown cells from the growth module to the filtrationmodule; 4) transfer the filtered cells from the filtration module to thetransformation module, 5) transfer the transformed cells to the samplewell, and 6) transfer the cells from the collection substrate to avessel without user intervention.
 12. The method of claim 11, whereinthe fluid immiscible with the cells in the aqueous medium is decane. 13.The method of claim 11, wherein the generated aqueous droplets comprisecells in a Poisson distribution.
 14. The method of claim 11, wherein thecells are bacterial cells.
 15. The method of claim 11, after the poolingstep, filtering the edited cells in the filtration module.
 16. A methodfor isolating and editing cells in the automated stand-alonemulti-module cell editing instrument of claim 2, comprising the stepsof: providing live cells in the receptacle configured to receive thelive cells; providing editing nucleic acids the receptacle configured toreceive editing nucleic acids; growing the live cells in a growth moduleto a desired optical density to produce grown cells; filtering andrendering electrocompetent the grown cells to produce filtered cells;transforming the filtered cells in a transformation module configured tointroduce the editing nucleic acids into the filtered cells to producetransformed cells; generating droplets in the microfluidic device byproviding the transformed cells in an aqueous medium in the sample well;providing the fluid immiscible with the cells in the aqueous medium inthe carrier fluid well; flowing the immiscible fluid from the carrierfluid well through the carrier channel to the channel intersection;flowing the cells in aqueous medium from the sample well through thesample channel to the channel intersection; generating aqueous dropletsin the immiscible fluid; and collecting the aqueous droplets one at atime in wells in the collection substrate; incubating the aqueousdroplets in the collection substrate to allow the editing nucleic acidsto edit the transformed cells; monitoring cell growth in the dropletsvia the detection station; sorting the aqueous droplets based onrapidity of cell growth; pooling the aqueous droplets with slow-growingcells in a vessel; and using an automated liquid handling system to 1)transfer the editing nucleic acids from receptacle configured to receivenucleic acids to the transformation module, 2) transfer the live cellsfrom the receptacle configured to receive the live cells to the growthmodule, 3) transfer the grown cells from the growth module to thefiltration module; 4) transfer the filtered cells from the filtrationmodule to the transformation module, 5) transfer the transformed cellsto the sample well, and 6) transfer the cells from the collectionsubstrate to a vessel without user intervention.
 17. The method of claim16, wherein the fluid immiscible with the cells in the aqueous medium isdecane.
 18. The method of claim 16, wherein the generated aqueousdroplets comprise cells in a Poisson distribution.
 19. The method ofclaim 16, wherein the cells are bacterial cells.
 20. The method of claim16, after the pooling step, filtering the edited cells in the filtrationmodule.